Abstract: The nuclease P1 was purified to obtain pure component by activated carbon decolorization, (NH₄)₂SO₄ precipitation, desalination and gel chromatography and its enzymatic properties was investigated. This purified enzyme had a specific activity of 33967 U/mg protein after 8.48-fold purification. The Michaelis constant (K_m), the maximum reaction rates (V_m) and the catalytic constant (K_cat) of the purified enzyme were 2.50 mmol/L, 0.0864 mmol/(mL·min) and 252.43 s⁻¹, respectively. The optimization pH and temperature for the nuclease P1 were at pH5.5 and 75 ℃. The enzyme was stable in the temperature range from 60 to 75 ℃ and in the pH range from 4.0 to 6.0. Zn²⁺ had a positive effect on the enzyme activity, while Cu²⁺ was a strong inhibitor of nuclease P1, Ni²⁺、Fe²⁺、Mn²⁺ had the different inhibition. This research laid a scientific foundation for the extensive application of the enzyme.