Abstract: Existing studies have revealed that the free calcium content in the supernate of fermented fishbone meal with lactic acid bacterial (LAB) significantly increases. In order to improve the fermentation technique, this paper investigated the optimum fermentation conditions for tilapia fishbone meal with a series of experiments. The results of single factor experiments indicated that the optimum LAB species was Lactobacillus casei, and the optimal ferment liquid volume was 30%, fishbone meal to liquid ratio was 1/5, optimal saccharide was glucose with natural pH value. The mixed level orthogonal test showed that the sequence of influence intensity to the supernate free calcium content of the factors were: fermentation period > glucose dosage > inoculation amount > temperature > grain size of fishbone meal, in which the fermentation period significantly affected the free calcium content in the supernate (p<0.05). The optimal fermentation conditions were: fermentation period 8 days, inoculation amount 8%, glucose amount 5%, temperature 37℃, and fishbone meal grain size < 0.075 mm.