To explore the function of gene SOST in intermuscular bone ossification of Carassius auratus in Qihe River, a modified whole bone staining method was firstly used to investigate ossification development of intermuscular bone. Then, the qRT-PCR, Western Blot and immunohistochemistry methods were employed to detect the mRNA and protein expression of gene SOST in different ossification stages. The results showed that the ligaments were found in myoseptum at 24 dpf (day post fertilization). The earliest epineural bones appeared at 25 dpf, and the epipleural bones were subsequently found at 28 dpf. At 33 dpf, the epineural bones began to bifurcate. Until 45 dpf, all intermuscular bones were fully developed in myoseptum. The gene SOST was expressed in various ossification stages of intermuscular bone and the lowest was at 23 dpf (P < 0.05). The mRNA abundance of gene SOST was increased along with the ossification development and reached a maximum level at 45 dpf (P < 0.05). The relative expression level of SOST protein at 45 dpf was significantly higher than at 23 and 27 dpf (P < 0.05), but had no obvious differences with other ossification stages (P > 0.05). The expression of gene SOST protein showed the similar trend with the mRNA and both of them varied along with the development process of intermuscular bone, which was at the lowest point at 23 dpf and climbed to the highest level when all the intermuscular bones appeared. Thus, we concluded that there were relationships between the gene SOST and ossification development of intermuscular bone, and the gene SOST may play an important role in morphology and ossification of intermuscular bone.