Objective To evaluate the health hazard of occupational exposure to anti-tumor drugs in the staff of pharmacy intravenous admixture services (PIVAS) by using environmental and biological detection methods.Methods In April 2020, 50 staff members were randomly selected from PIVAS of our hospital as the study subjects. They were divided into exposed group (25 cases) and non-exposed group (25 cases) according to whether they had been exposed to anti-tumor drugs or not. Taking 5-fluorouracil (5-Fu) and cyclophosphamide (CTX) as detection markers, the concentrations of 5-Fu and CTX in the blood and urine samples of subjects in two groups, as well as inthe wipe samples of different objects in PIVAS, were determined by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS) for two consecutive years (2020 and 2021). The apoptosis rate of peripheral blood cells was measured by flow cytometry. The level of 8‐hydroxy‐2'‐deoxyguanosine(8-OHdG) was determined by enzyme-linked immunosorbent assay.Results There was no statistical difference in the general situation of the staff. The surface of the objects inPIVAS environment was polluted by 5-Fu and CTX in different degrees. The average concentration of 5-Fu on the surface of oscillator, transfer bin handle and telephone in the configuration area was 2 286.26, 976.82 and 202.42 ng?mL^-1 respectively, and the average concentration of CTX was 2 991.26, 88.12 and 155.40 ng?mL^-1, respectively. CTX residues exist in 5-Fu and CTX storage tanks. The results of flow cytometry in 2021 showed that the early and total apoptosis rates of monocytes both were higherin the exposed group than in the non-exposed group (P=0.024, P=0.024). The early and late apoptosis rates of neutrophils in the exposed group both were higher than in the non-exposed group (P=0.004, P<0.001). CTX residues were detected in urine of the staff in both years, and the detection rate of urinaryCTX of the exposed group was higher than that of the non-exposed group (χ²=11.52, P<0.001). The plasma concentration of CTX in the exposed group was higher than that of the non-exposed group [Z=-7.500, P<0.001 (2020); Z=-4.500, P<0.001 (2021)]. 8-OHdG was recognized as a sensitive biomarker for evaluating DNA damage and oxidative stress. The level of 8-OHdG in the exposed group was higher than that of the non-exposed group (Z=-4.605, P<0.001), indicating that the degree of DNA damage in the exposed group was significantly higher than that in the non-exposed group.Conclusion PIVAS is polluted by anti-tumor drugs to a certain extent, so we should pay attention to the impact of occupational exposure to anti-tumor drugs on human health, and strengthen protection as far as possible.