Abstract: Visible spectrophotometry was applied to the determination of kanamycin sulfate with ninhydrin as chromogenic agent, based on the chromogenic reaction of kanamycin sulfate with ninhydrin to form a complex with characterized adsorption at 563 nm. The optimum conditions for chromogenic reaction were defined by investigating the conditions including the acidity and dosage of buffer solution, the dosage of ninhydrin solution, reaction time through heating and sample order. A linear relationship was found between the absorbance and the mass concentration of kanamycin sulfate in the range of 0.074-0.465 g·L-1 with detection limit (0.01/k) of 0.026 g·L-1. Recovery rates obtained by standard addition method were in the range of 95.5%-98.4%, and RSDs (n=12) were less than 3.0%.