Abstract: To screen and optimize the key process parameters for probiotic fermentation of Muraenesox cinereus bone, break through the technical bottleneck in their high-value utilization, address the issues associated with the limited and extensive traditional utilization methods of M. cinereus bone, as well as expand the variety of its refined processing products, we employed Bifidobacterium animalis, Lactobacillus rhamnosus, and Lactococcus lactis to ferment M. cinereus bone. The soluble calcium content in the product was measured using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to identify the optimal probiotics. Response surface experiment was designed with the soluble calcium mass concentration in the fermentation broth serving as the response variable, to screen the predictive model and determine the optimal parameters of the fermentation process. Scanning Electron Microscopy (SEM) was utilized to analyze the attachment of probiotics to M. cinereus bone. The results indicate that L. rhamnosus was the optimal fermentation probiotics for soluble calcium dissolution from M. cinereus bone, with the Least Squares Model identified as the best model, achieving an r² of 0.901 7 and a relative average square error (RASE) of 6.064. The optimal fermentation conditions for L. rhamnosus were determined as follows: temperature of 35 ℃, bacterial inoculation concentration of 10⁷ CFU·mL⁻¹, and fermentation time of 41 h. Under these conditions, the mass concentration of soluble calcium in the fermentation broth was (285.25±2.29) mg·L⁻¹. SEM analysis further confirms that L. rhamnosus showed optimal growth within the fermentation matrix containing M. cinereus bone. This study provides a technical reference for the development of probiotic calcium products using M. cinereus bone waste.