Aim To establish a method of cystathionine β-synthase activity assay and detect cystathionine β-synthase activity in various tissues of rat. Methods We first extracted cystathionine β-synthase from various tissues of rat. And then the final concentration of the assay components in 0.4 mL of incubation mixture were: 100 mmol/L Tris-HCl (pH 8.6), 0.25 mmol/L pyridoxal 5'-phosphate, enzyme protein (2.5～40 mg), 20 mmol/L serine, and 20 mmol/L L-homocysteine. After incubation for 60 min at 37℃, the reaction was stopped by addition of 0.05 mL 50% trichloroacetic acid. Following 5 min incubation on ice, the precipitated protein was removed by centrifugation. To a 0.2 mL aliquot of a supernatant, 3.3 mL of ninhydrin reagent was added and the color was developed. The absorbance at 465 nm was read against an substrate and enzyme-free blank was subtracted. Results Colorimetry to detect the cystathionine β-synthase activity was established; and cystathionine β-synthase activity in various tissues of rat was different. Conclusion The method established can detect cystathionine β-synthase activity successfully. It had the merit of high precision and good repetition and was easy to operate.