Bio-calcium derived from fish frames may offer several advantages for osteoporosis prevention. This study aimed to evaluate the effects of bio-calcium derived from skipjack tuna frames on bone loss in ovariectomized rats. Tuna bio-calcium was prepared through enzymatic hydrolysis, defatting, bleaching, and grinding processes. The bioavailability of calcium was tested using the Caco-2 cell monolayer model, showing that 13% of tuna bio-calcium was absorbed, compared to 10% for calcium carbonate. Rats were divided into the five following groups: (1) OVX, (2) sham-operated, (3), OVX + estrogen-treated (4) OVX + calcium carbonate-treated, and (5) OVX + tuna bio-calcium-treated. All groups were raised for eight weeks. Tuna bio-calcium was able to increase BV/TV by 26% in the femur and 29% in the tibia, compared to 13% and 17% in the OVX group, respectively. Trabecular thickness in the femur upsurged to 360 µm in the tuna group, while a thickness of 290 µm was observed in the control. Additionally, osteoclast numbers were reduced to 5 N.Oc/mm in the femur and 6 N.Oc/mm in the tibia in the tuna group, compared to 35 and 45 N.Oc/mm in the control. Overall, tuna bio-calcium effectively prevented bone loss and can serve as a promising natural alternative for managing osteoporosis.