The Zhenyuan (镇沅) gold orefield is located in the Ailaoshan (哀牢山) gold belt, Sanjiang (三江) metallogenic region, China. The fluid inclusions (FIs) in mineralized quartz samples, collected from four deposits, i.e., Laowangzhai (老王寨), Donggualin (冬瓜林), Kudumu (库独木), and Daqiaoqing (搭桥箐), in the orefield, were studied to reveal ore-forming process. Only aqueous-type and H2O-NaCl-type FIs were observed in the samples. Two generations of FIs are identified due to the spatial occurrences of FIs in host minerals. The early generation is located in the internal zone of quartz crystal, and it has higher homogenization temperatures varying from 180 to 255 °C and salinities ranging from 8.7% to 11%; the late generation, often with beating bubbles under room temperature, is developed in the quartz margin, its homogenization temperatures is lower within 95 to 160 °C. After elimination of the late generation of FIs, early generation FIs compositions were tested. Gaseous phases in the FIs were H2O and CO2, with low contents of N2, CH4, and C2H6, and the liquids mainly contains Na+, K+, Ca2+, Mg2+, Cl−, and SO4 2−. The reappraisal of the previously published hydrogen and oxygen isotope results suggested that the early generation of ore-forming fluid was mainly primary magma hydrothermal, while the late generation fluid contains large amounts of meteoric water. Thus, the result showed that Zhenyuan gold orefield may differ from typical orogenic deposits, which contains abundant CO2-dominated FIs. The two generations FIs developed as quartz grew displaying a continuous evolution of the ore-forming fluids.