To establish the simple,accurate and inexpensive determination method of yeast RNA hydrolyzed yield.Method: The four single nucleotides of RNA hydrolysate were determined by the combined methods of paper electrophoresis and paper chromatography;the precision and accuracy of this method were analyzed using coefficient of cariation(CV) and average recovery.Result: Combined the 10% acetic acid buffer and the saturated(NH4)2SO4:1M ammonium acetate:isopropanol(80:18:2,V/V);developing solvent,the four single nucleotides could be separated simultaneously;the relative coefficient(R) were all more than 0.985;the CV value of AMP,GMP,CMP,UMP was 1.30%,1.44%,1.36% and 1.30% respectively in the standard nucleotides mixture sample,while the CV value was 2.45%,2.01%,2.75% and 2.06% respectively in the yeast RNA hydrolization liquid;the average ratio of recovery were between 94.80% and 106.57%;the minimum detection limit were between 0.65~0.72mg/mL.Conclusion: The determination method deployed in this paper had the advantages of meticulous precision,high degree of accuracy,lower investment of instrument and cheaper cost,so it can be promoted widely.