Myoglobin (Mb) is the main constituent of vertebrate skeletal muscle and myocardium and plays an essential role in oxygen binding, storage, transport, and earliest disease diagnosis. This study focuses on preparing the novel recombinant rabbit anti-Mb monoclonal antibody and applying it to a diagnosis of Mb deposition in rhabdomyolysis-associated acute kidney injury (RM-AKI). The full-length coding sequence of rat Mb was cloned and expressed, and the high-quality and titer rabbit anti-Mb polyclonal antibodies were produced by the immunogen His-Mb fusion protein. A new hybridoma cell was obtained by hybridoma screening technology. With the help of DNA sequencing and a molecular clonal, anti-Mb monoclonal antibody heavy and light chains expression plasmid was constructed. Finally, the recombinant rabbit anti-Mb monoclonal antibody with extraordinarily high affinity (KD= 1.21 pM) was obtained. Meanwhile, it had broad species reactivity (mouse, rat, human, and horse) and good tissue specificity (skeletal muscle and myocardium). It also had a very good performance in western blotting, immunohistochemistry, and immunofluorescence assay to detect the Mb level in the kidney, myocardium, and skeletal muscle of RM-AKI. This study will be significantly helpful for Mb-associated disease diagnosis, and pathogenesis exploration, and further may act as a neutralizing antibody for disease treatment.