Abstract: Objective：To establish rate method for determination of seminal plasma fructose and evaluate its performance. Methods：The fructose concentration in seminal plasma was determined by rate method and the detecting parameters for the automatic biochemical analyzer were set up. The reagent blank absorbance, accuracy, repeatability and linear range of the automatic method were evaluated and the results of the method were compared with those of indole chromogenic method commonly used in clinical laboratories. Results：The average absorbance of the reagent blank was 0.032 and the average change rate of blank absorbance (△A/min) was 0.000 2. The coefficients of variation (CV) in 10 repeated determinations for 3 seminal plasma samples with high, middle and low fructose concentration, were 1.40%, 1.57% and 2.79%, respectively. The recovery rates ranged from 95.77% to 100.97%. A good linear relationship (r²=0.997 8) was shown when the concentration of seminal plasma fructose was between 5 and 35 mmol/L. Compared with the results of indole chromogenic method, the rate method showed no statistical difference (［20.92±8.62］ mmol/L vs ［21.15±8.37］ mmol/L, t=-1.157, P=0.250) and the results of 115 seminal plasma samples showed significant positive correlation ((r²=0.939 5, P<0.01) between the two methods. Conclusion: The established rate method in the determination of seminal plasma fructose concentration showed low reagent blank, good repeatability and accuracy, and fine concordance with the colorimetric method used routinely in clinical laboratories. The method should be simple, rapid, and suitable for screening large numbers of samples, and greatly saves the resources of manpower and reagents. The dilution of seminal plasma samples is unnecessary for the determination.