Sperm densitometer has been widely applied in mammals, whereas little research has been done in poultry. Taking Black Silkies chicken and Youxian Partridge Duck semen as experimental materials, the sperm densitometer and blood cell counting board were used as testing tools, the factors affecting the determination accuracy rate were analyzed, and the roosters and drakes sperm absorbance-density curve were established and verified. The results showed that the absorbance should be measured as soon as possible after the semen was diluted using a diluent of 3%NaCl. There was great influence on the absorbance measurement when the original semen was diluted by different mixing method. The absorbance of B liquid is significantly higher than that of other 3 groups(P＜0.05)when the same semen was diluted 10 times with 3%NaCl and other three kinds of common semen dilutions, and there were no significant differences within the other 3 groups (P>0.05). Using the 3.0%NaCl as diluents, the relationship of the absorbance-density curve was cubic function for the rooster and ducks semen, and the regression equations were Y1=1.374X3-0.786X2+0.945X-0.002(R2=0.997)and Y2=1.283X3-0.899X2 +0.994-0.009(R2=0.996).It was feasible to simplify the sperm density determination methods using 0.9%NaCl instead of 3%NaCl, and the regression equations was Y3=-0.264X3+1.23X2+0.468X+0.019 (R2=0.999). The optimal range of absorbance for the curve of roosters semen 0.9%NaCl and 3%NaCl was between 0.035~0.692 and 0.069~0.624. And as for 3%NaCl drakes semen, it might be less than that of 3.0%NaCl roosters semen. There was no significant difference between the cell count and the curve calculated density within each optimized range (P>0.05). The average relative error of the 0.9%NaCl and 3%NaCl was 6.95%and 3.11%when using the counting value as the true one which is within each optimized absorbance range, and that of 3%NaCl darkes semen was 4.61%with the absorbance range of 0.054~0.123. This paper will promote the research and development of hand-held sperm densitometer for poultry and better use of mammalian sperm densitometer.