In this experiment, response surface methodology was used to study the preparation of malic acid calcium salt from bovine bones assisted by ultrasonication. The results showed that the optimum conditions for ultrasound-assisted preparation of calcium malate from bovine bone were as follows: solid-liquid ratio 1:15, solid-acid ratio 1:1.5, ultrasonic power 200 W, ultrasonic temperature 35°C, and ultrasonication time 17 min. The efficiency of calcium recovery was 66.16%, and the purity was 92.54%. After three ultrasonic treatments of 17 min each, the calcium malate conversion rate of bovine bone reached 95.73%. Animal experiments showed that feeding bovine bone-derived calcium malate significantly increased alkaline phosphatase (ALP) activity and bone calcium content, reduced tartrate-resistant acid phosphatase (TRAP) activity, and maintained the balance of serum calcium and phosphorus. These results indicated that the ultrasonic method effectively ionized calcium in bovine bone, which provides a reference point for the industrial production of calcium products with bovine bone as the raw material.