Abstract: The liquid-liquid phase separation (LLPS) of proteins and nucleic acids plays an important role in the formation of membrane-free organelles. In pathological studies, the LLPS can also lead to the abnormal accumulation of proteins and lead to some neurodegenerative diseases. Therefore, the study of the effective regulation of the LLPS of protein-nucleic acid can provide some reference value for us to understand the biological structure and function and the treatment of related diseases. Optical microscope, dynamic light scattering and ultraviolet spectrophotometers were used to study the regulation of the LLPS of poly-L-lysine (PLL)-deoxyribonucleic acid (DNA) by monovalent/divalent cations. The formation mechanism of LLPS phenomenon of PLL-DNA was also analyzed. It was observed that the concentration of sodium/potassium ion was higher than 100 mmol/L, the LLPS of PLL-DNA precipitated gradually and precipitated mostly at 600 mmol/L. When the concentration of sodium/potassium ion was higher than 600 mmol/L, the polymer precipitation began to dissolve, and LLPS occured again. However, magnesium/calcium ion showed a higher efficiency in the regulation of PLL-DNA polymers. The critical concentration of magnesium/calcium ion to form precipitation was 50 mmol/L, and the concentration of re-occurrence of LLPS was about 300 mmol/L. The absorbance of the solution was analyzed and the same conclusions were obtained as observed in the microscopic experiment. The electrophoretic mobility of the solution was reversed that the value change from negative to positive with the increase of the concentration of monovalent/divalent cations, indicating that the regulation of monovalent and divalent cations on protein-nucleic acid LLPS is related to the change of charge, and the regulation by divalent cations is also more efficient than monovalent cations. Finally, a structural model of the polymer was proposed which provides a reasonable explanation for the phase separation process.