As a giant actin-binding protein with molecular weight of approximately 500-900 ku, nebulin is thought to act as a molecular template that regulates the length of thin filaments and plays an essential role in the assembly and maintenance of I-Z-I bands in myofibrillar muscles. Though nebulins from mammals have been studied widely, much less study was performed on nebulin from fish muscle. In the present study, nebulin from the skeletal muscle of sea bream (Sparus latus) was first extracted with low ionic strength buffer, followed by applying to Sephacryl S-400 gel filtration chromatography and finally purified by electrophoretic elution. Purified nebulin revealed a single protein band on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), suggesting its high homogeneity. A polyclonal rabbit anti-nebulin antibody was thus prepared by immunizing rabbit with purified nebulin and immunoglobulin G (IgG) was purified by Protein A-Sepharose affinity column. The immunogeneicity of the purified antibody was further identified by Dot-Blot and Western-Blot. The potency of anti-nebulin antibody reached 5×10⁴ by Dot-Blot analysis. Western-Blot revealed that the antibody immunologically reacted with purified nebulin and nebulin in myofibrils while no cross reaction with other myofibrillar proteins was detected, suggesting the high specificity of the polyclonal antibody. Thus, the antibody prepared in the present study will surely beneficial for further study concerning the detection of this protein immunologically. The degradation of nebulin during fish storage at two temperatures of 4 ℃ and 18 ℃ was also investigated by SDS-PAGE. At 4 ℃, protein degradation could be detected after storage for 9 d and after 18 d, more than 70% of the nebulin original band disappeared. However, at 18 ℃, nebulin deterioration could be detected even at 24 h and after 120 h, the original protein band disappeared completely. The decomposition of nebulin as detected by SDS-PAGE was in accordance with sensory freshness change, suggesting the completeness of nebulin is an index of fish muscle freshness. Interestingly, the largest protein (titin) almost did not change even after storage at 4 ℃ for 18 d or at 18 ℃ for 188 h. In conclusion, our present study, especially the anti-nebulin antibody prepared provided an effective tool for the investigation of fish freshness.