Besides cultivation, extraction is also a critical stage in enhancing the yield of phycocyanin production - a highly valuable compound from Spirulina biomass. In this study, the combined effect of three important variables in the ultrasonic-assisted extraction process on phycocyanin extraction yield, namely extraction temperature, sonication time, and solvent pH were investigated through a central composite design experiment. Furthermore, the response surface method was applied in order to define an optimal condition to achieve the highest extraction yield. The results showed that when temperature ranged from 35ºC to 45ºC, sonication time from 20 to 50 minutes, and solvent pH from 6 to 8, the average yield of 30.135±1.552 mg/g was obtained with an average purity of 0.871±0.043. A regression model was also successfully developed, which allowed a good prediction of extraction yield based on the three mentioned variables. On the other hand, an optimal condition for extraction was also proposed with sonication time = 43.57 minutes, extraction temperature = 37.6oC, and solvent pH = 6.7. These results were practically valuable for the improvement of phycocyanin extraction from Spirulina biomass.