The Tianluoshan Site of Yuyao City in Zhejiang Province is a typical Neolithic site with a complete preservation of the Hemudu culture (ca. 7–5 ka). This culture is well-known for its important role in studies on early rice domestication. Studying samples from the Tianluoshan Site provides a good opportunity to understand regional paleoenvironmental changes and their impact on Neolithic settlement. In this study, 263 cm samples were obtained from the T005W profile in the Tianluoshan Site Museum. The profile samples were subjected to lithologic stratigraphic description, accelerated mass spectrometry (AMS)14C dating, high-resolution X-ray fluorescence (XRF) spectrometry, elemental scanning (2 mm/sample), diatom (20 cm/sample) and particle size analysis (2 cm/sample). The paleoenvironmental changes reflected by this profile could be divided into three stages based on multi-proxy analysis. (1) In the first stage (263–211 cm, about 7.0–6.5 cal ka BP), or the first and second cultural periods of Hemudu, the cultural layer was stacked and mixed with high contents of sand, gravel, and pottery pieces, thereby showing obvious disturbance from human activities. The diatom assemblage was dominated by aerophilous species, such as Hantzschia amphioxys , which live in shallow swamp. These findings indicated that this period was dominated mainly by a freshwater swamp environment. The geomorphic condition provided Neolithic people with a suitable environment to colonize and develop rice cultivation. (2) In the second stage (211–28 cm, around 4.2–4.0 cal ka BP), the layer had a thickness of approximately 180 cm. However, five AMS 14C dates from this stage were between ca. 4.2 and 4.0 cal ka BP, and the lithology of these overlying strata were inconsistent with the cultural layer underneath. As such, a hiatus existed between the two stages. The lithological feature of this stage mainly consisted of a parallel layer with a couplet of interbedded clay silt and white-gray sand layer, which represented deposition under hydrostatic conditions after flooding. This part also contained multiple yellow oxide layers, possibly indicating transient exposure. The intensity of Ca between 211 and 166 cm was low, but this value increased rapidly above 166 cm and stabilized at a higher level than that of the first period. The dominant diatom assemblage changed from a freshwater group ( H. amphioxys ) at the bottom of this part to a marine and brackish group (e.g., Cyclotella striata var. striata and Actionptychus undulates ). In this period, the rapid deposition of flooding events likely occurred as initiated by heavy precipitation (211–166 cm), which was indicated by low Ca and freshwater aerophilous diatom assemblage. Afterward, multiple storm surges resulted in sedimentation (166–50 cm) with marine-brackish characteristics. (3) In the third stage (28–0 cm, no date), the layer was characterized by a mixture of brown clay, silt, sand, and gravel with low contents of water, iron, and manganese oxide spots. Ca was weak, and clay content was high. This period was mainly dominated by freshwater epiphytic diatom species, such as Pinnularia vividis and Navicula cuspidate , and affected by modern farming. This study revealed the background of the hydrological environment during the first and second cultural periods of the Tianluoshan Site and the flooding events around 4.2–4.0 cal ka BP, which were essential for the understanding of the environmental background of Neolithic people and the extreme regional events in the Tianluoshan Site. The stratigraphic deposits of different sections in the site varied because of the influence of microtopography. Therefore, studies on the paleoenvironment of the Tianluoshan Site should involve a systematic analysis of multiple sections and cores around this site.