The present study was designed to optimize the different conditions for b-cyclodextrin (b-CD) crosslinked by adipic acid on cholesterol removal from squid liver oil. Different factors were concentrations of crosslinked b-CD, mixing temperature, ratio of squid liver oil to distilled water, mixing time and mixing speed. It was found that cholesterol removal from squid liver oil was significantly affected by concentrations of crosslinked b-CD, mixing temperature, ratio of squid liver oil to distilled water, mixing time and mixing speed. In a recycling study, cholesterol removal from the squid liver oil with recycled crosslinked b-CD in the first recycling trial was 81.05%, which was slightly lower than that with new crosslinked b-CD (87.06%). In up to three time trials, over 70% of cholesterol removal was observed. The present study indicated that the optimum conditions for cholesterol removal from squid liver oil using crosslinked b-CD were a 1:3 ratio of squid liver oil to distilled water, 25% (crosslinked b-CD/distilled water, w/v) crosslinked b-CD concentration, 20 min mixing time, 800 rpm mixing speed and 55°C mixing temperature with about 87% cholesterol removal.