Abstract: The aim of the experiments was to optimize the determination of methionine (Met) in the fermentation broth by the sodium nitroprusside (SNP) method. The ultraviolet visible spectrophotometer was used to analyze the absorbance peak of different reaction,including different amino acids with SNP and Met with different reagents for different time. The optimal colorimetric procedure of SNP method was obtained with single factor tests,including the volume of glycine (Gly),the volume of NaOH,the amount of SNP added and the volume of concentrated phosphoric acid added,and response surface methodology was used to optimize the SNP method. The results showed that other amino acid had no affection on the determination of Met except for histidine (His) after reaction with SNP. The absorption peak was at 513 nm decided by the addition of phosphoric acid. The components of the broth had no new adsorption peak after reaction with SNP. The optimum determination method of Met with SNP was obtained by single-factor experiments and response surface method. The fermentation broth was centrifuged at 1500×g for 15 min,5 mL the supernatant was added 0.74 mL 5 mol/L NaOH and 0.15 mL 10% SNP solution;the tube was shaken and allowed to stand 30 min;2 mL concentrated phosphoric acid was added to the mixture drop by drop. Then the tube was reacted shakingly for 5 min,and measured at 513 nm The value of absorption of 1 g/L Met was 1.952 with optimized SNP method. It was confirmed by spectral analysis that the addition of Gly could reduce the peak absorption of Met and His reaction with SNP method. It was possible to determine whether His affected the determination of Met by analyzing whether the maximum absorption wavelength was at 513 nm. The SNP method could be used to screen methionine producing bacteria quickly.