OBJECTIVE：To establish the method for simultaneous determination of 5-fluorouracil（5-FU）and its metabolites 5-fluoro-5，6-dihydrouracil （5-FUH2） in human plasma ，and apply it in the clinic. METHODS ：After plasma samples were processed twice by ethyl acetate ，UPLC-MS/MS method was adopted using 5-bromouracil （5-Bru） as internal standard. The determination was performed on Acquity UPLC HSS T 3 column with mobile phase consisted of methanol-water （30 ∶ 70，V/V）at the flow rate of 0.3 mL/min. The column temperature was 20 ℃，and sample size was 5 μL. An electrospray ion source was used to carry out negative ion scanning with multiple reaction monitoring. The capillary voltage was 1.5 kV；the taper hole voltage was 20 V；the desolvent temperature was 450 ℃；the desolvent air flow was 850 L/h；the cone hole gas velocity was 50 L/h. The ion transitions for quantitative analysis were m/z 129.00→41.90（5-FU），m/z 130.87→82.92（5-FUH2），m/z 189.00→42.10（5-Bru）， respectively. From Aug. to Oct. 2020，10 patients with advanced colorectal cancer were treated with continuous intravenous drip of 5-FU for 46 hours were collected from Harbin Medicinal University Cancer Hospital. Steady-state plasma concentration of 5-FU and plasma concentration of 5-FUH2 were determined at 18-30 h of continuous intravenous drip. The area under the curve （AUC）for 5-FU and concentration ratio of 5-FUH2/5-FU were calculated. RESULTS ：The linear range of 5-FU and 5-FUH2 were 20 to 1 000 μg/mL（R 2＞0.990）. The quantification limits were 20 ng/mL. RSDs of precision test were all lower than 20％，and relative error ranged ±10％. The extraction recovery and matrix effects didn ’t affect the determination of substance to be measured. Among 10 patients with advanced colorectal cancer ，the steady-state concentration of 5-FU were 180.04-622.83 ng/mL，and AUC of 5-FU ranged from 8.28 to 28.65 mg·h/L. The concentration of 5-FUH2 ranged 336.48-948.43 ng/mL，and concentration ratio of 5-FUH2/ 5-FU ranged 0.93-4.21. AUC of 5-FU in 10 patients had about 3-4 fold individual differences. CONCLUSIONS ：The established method has good precision and accuracy ，high sensitivity ，and simple operation. It can be used for plasma monitoring of 5-FU in patients with advanced colorectal cancer.