Bixin is a principal color component of annatto pigment which is obtained from the seed coat of Bixa orellana L., a tropical shrub. The extraction yield of bixin from annatto seeds using acetone, soybean oil and sodium hydroxide solution with different extraction conditions were investigated in this study. The extraction time, temperature, solid-liquid ratio and light exposure showed significant effects on the bixin yield. The extraction using soybean oil had the lowest bixin yield followed by the extraction using sodium hydroxide solution. The extraction using acetone resulted in the highest extraction yield (68.1%) after only 40 min. The extraction using soybean oil at 100oC led to two-fold bixin yield compared to that operated at 80oC while the mild temperature (50oC) was found to be the most suitable for the extraction using sodium hydroxide solution. The exclusion of light exposure by covering extraction beakers with aluminum foil could significantly improve the bixin extraction yield due to the reduction in bixin degradation. Therefore, the extraction using acetone avoiding light exposure is suggested for recovering bixin from annatto seeds.