Zebrafish are widely used in mechanistic studies related to bone degradation and during new drug development. At present, the clinical application methods of bone mineral density (BMD) mainly include single-photon absorption, two-photon absorption, dual-energy X-ray absorption, quantitative CT, and ultrasonic technology. All these methods are difficult to use in zebrafish. This article aims to seek a new, applicable and easy-to-operate method for quantitative analysis of zebrafish bone mineral density. The optical coherence tomography images of zebrafish with 125 μmol/L prednisolone solution induced osteoporosis at 0, 7, 14, and 21 days were sequentially grayed, binarized, image segmented, and threshold segmented, and were done density projection, and finally bone density statistics. The experimental results showed that this method can effectively and quantitatively analyze the degree of osteoporosis in zebrafish on different days. This method is suitable for studying the effects of different exercise modes on zebrafish bone mineral density during the growth period, high-throughput zebrafish osteoporosis drug screening, drug evaluation, etc. It is a new method for quantitative analysis of zebrafish bone mineral density.