Folic acid is an important food source vitamin. In order to improve the efficiency of folic acid determination， a system and method for the determination of folic acid by on-line photocatalytic oxidation derivatization combined with fluorescence spectrophotometry based on microtubular reactor was developed. System operating parameters were optimized， and the method was investigated and evaluated， then applied to the determination of actual samples. The results showed that nano-titanium dioxide catalyst was successfully corroded and coated in spiral tube quartz microreactor with small inner diameter， and folic acid on-line derivatization was realized by photocatalytic oxidation with ultraviolet light irradiation. The optimal derivatization pH was 4.2， flow rate was 250 μL/min， the maximum excitation and emission wave length were 280 nm and 442 nm. Under the optimal conditions. There was a good linear relationship between fluorescence intensity and folic acid concentration in the range of 0.1-2.0 mg/L. The standard curve equation was F=381.68c+10.53， the correlation coefficient and detection limit (3s/k) was 0.998 and 7.2 μg/L， respectively. The relative average deviation of operation stability was 0.9%-2.4%， meanwhile， it has excellent anti-interference ability to a variety of common substances. When the method was applied to folic acid tablets， it exhibited a relative error was less than 5%， the recovery rate was between 94.7% to 102.1%， and the relative average deviation was less than 2.1%.