In this study, a steam explosion pretreatment method was established to prepare tuna bone powder. The conditions were optimized such that steam pressure of 0.6 MPa, reaction time of 5 min, and sample weight of 100 g. The result showed that steam explosion pretreatment would not change the chemical structure of bone powder, however, the median particle size (D) of the steam explosion pretreated tuna bone powder (SE-TBP) (13.186 μm) was significantly smaller than that of normal biological calcium tuna bone powder (N-TBP) (169.762 μm). The calcium absorption rate (79.75 ± 2.33%) and utilization rate (78.75% ± 2.85%) of the mice fed with SE-TBP were both higher than those of fed with CaCO or N-TBP with the same calcium equivalent in the feed. The steam explosion pretreatment method could obtain ideal tuna bone powder in a shorter time, provide a method for deep processing and utilization of tuna bone by-product.