With the wide application of nanomaterials, the concentration of nanomaterials in natural water continues to increase, which poses a severe threat to the water environment. However, the influence of organic matter and nanomaterials rich in natural water on the toxic effect of algae growth is still unclear. In this study, the effects of humic acid (HA) and nano-cerium oxide (nCeO2) on the physiology and transcriptome of Chlorella sp. were analyzed, and the mechanism of the toxic effect of HA on Chlorella sp. under nCeO2 stress was revealed. Under 20-200 mg/L nCeO2 stress, the growth of Chlorella cells was inhibited and the highest inhibition rate reached 52% within 200 mg/L nCeO2. The Fv/Fm and ETRmax values of Chlorella sp. decreased from 0.490 and 24.45 (20 mg/L nCeO2) to 0.488 and 23.4 (100 mg/L nCeO2), respectively. Under the stimulation of nCeO2, the level of reactive oxygen species in algal cells was increased, accompanied by lipid peroxidation and membrane damage. However, the addition of HA at concentrations of 5-10 mg/L effectively alleviated the toxic effect of nCeO2 on Chlorella sp. Transcriptome analysis showed that 10 mg/L HA could alleviate the cellular stress at 100 mg/L nCeO2 on Chlorella sp. by regulating genes related to photosynthesis and metabolism pathways. Moreover, the downregulation of genes (e.g., Lhca1, Lhcb1, AOC3, and AOC2) indicated that HA reduced the level of oxidative stress in Chlorella sp. These findings offer novel insights of evaluating the ecotoxicity nCeO2 and HA in natural water environment and their impact on Chlorella sp.