The invention discloses a preparation method of a recombinant myoglobin. The method comprises that firstly, porphyrin is caused to be separated to prepare apomyoglobin by protein denaturation under anacidic condition of pH=2.0, and then the recombinant protein is synthesized by combining apomyoglobin renaturation with zinc porphyrin under an alkaline condition of pH=9.5. The preparation method issimple and easy to control. The prepared recombinant myoglobin can emit stable and strong fluorescence to myoglobin, and the fluorescence intensity is much stronger than that of protomyoglobin. The recombinant myoglobin can be used for preparing fluorescent biosensors, has the advantages of high sensitivity and good selectivity, and can be applied in biomedical research, environmental monitoringand other fields.