Summary The research was aimed to study the impact of various treatments on characteristics of bio‐calcium from hybrid catfish bone. Bio‐calcium from hybrid catfish bones (raw bone) was prepared using several 4 steps including high‐pressure water jet process (washed bone), soaking in alkaline solution (protein removal), ethanol immersion process (lipid removal), bleaching and grinding to obtain bio‐calcium powder. The obtained bio‐calcium had a yield of 14.46 g 100 g−1. Bio‐calcium had low protein, fat and moisture contents but had high ash content (P ≤ 0.05). Bio‐calcium was rich in calcium and phosphorus. Bio‐calcium contained high hydroxyproline content of 27.70 mg g−1 samples. Moreover, high contents of proline, glycine, aspartic acid alanine and glutamic acid were found in bio‐calcium. The ethanol immersion, bleaching and grinding process effectively decreased Thiobarbituric acid‐reactive substances (TBARS) values and total volatile acids (P ≤ 0.05) of bio‐calcium powder compared with washed bone, protein and lipid removal processes. Therefore, this research provided an alternative source for bio‐calcium production, which can develop as fortified in food products, calcium supplements and biomedical applications.