Nuclease P1 gene (nuc P1) which was cloned from Penicillium citrinum and expressed in A. niger Bdel4 with the low-background extracellular protein. The expression strategy of multi-copy nuc P1 in the A. niger with the linker of 2A peptide was applied to improve the enzyme activity of nuclease P1, the highest activity up to 77.6 U/mL. After Ni-chelate purification, the specific enzyme activity, the optimum temperature and pH were 32.4 U/mg, 65 °C and 5.3 respectively. The recombination nuclease P1 was activated by addition of Mg, Zn and Cu, and inhibited by addition of Ca, Fe, Mn, Ni, Co, Mg, K and EDTA. Furthermore, the enzyme hydrolyses yeast RNA efficiently into 5'- nucleotides. Through enzymolysis, the highest concentration of nucleotides achieved 15.12 mg/mL, and 75U nuclease P1 is suitable amount should be added to the enzymolysis system.