The invention discloses a method of separating nucleotide acid from enzymolytic liquid. The method particularly includes: at room temperature, adjusting pH of the enzymolytic liquid to 6-8; pumping a solution into a dilution chamber of an electrodialysis device; pumping a certain amount of purified water into a concentration chamber; after sufficient circulation, connecting a cathode and an anode of the electrodialysis device with a cathode and an anode of a direct-current power source; setting voltage at two ends of a membrane stack to 45 V or constant current to 0.5 A, and starting the electrodialysis device; in the electrodialysis process, using sodium hydroxide to maintain pH of a dilution chamber solution to greater than 6.0, and when electroconductivity of the dilution chamber solution is less than 0.1 ms/cm, stopping electrodialysis. A nucleotide solution high in purity is obtained in the concentration chamber, light transmittance of the nucleotide solution below 430 nm is about 70-90% and is increased by higher than five times than that of raw material liquid, and total yield is greater than 95%. Total nucleotide left in the dilution chamber is less than 2%.