Abstract: Nostoc sphaeroides is well-known for its edible and medical characteristics in cyanobacteria, particularly such as those produced in Enshi, Hubei Province. However, in pace with the changes in times and environmental condition, further development of the resource is required based on special gene or molecular activities, such as the heme oxygenase, HO1, which catalyzes heme degradation and positively roles in tissues by reducing oxidative injure, inhibiting apoptosis and so on. But in N. sphaeroides, the gene for encoding HO1 has not been clear up to today. Here, the cloning of HO1 (heme oxygenase) gene from N. sphaeroides, and prokaryotic expression the protein, analysis and alignment of amino acid sequences and molecular evolution, structural simulation by Swiss-model advanced have been perfused in order to clarify the molecular evolutionary status and advanced structure of the gene coding protein. The PCR results showed that the coding region of the gene Ns-HO1 was 678 bp, encoding a protein of 225 amino acids; sequence analysis indicated that amino acid similarity between Ns-HO1 and the homologous proteins reached 90.3%. Amino acid sequence alignment showed that there were a few replacement mutations in the amino acids, but the prebinding sites (e.g., Arg10, Tyr125) for the binding sites of the enzymatic substrate heme and metal ion (His17) and other key sites were maintained. A recombinant expression vector was constructed, and the Ns-HO1 gene was successfully induced in E. coli, SDS-PAGE detected the target protein at 26.0 kD in MW size; molecular phylogenic tree showed that Ns-HO1 was clustered in the common clade with the homologous protein from the N. flagelliforme and N. commune, and they all shared the closest common ancestor with the homologous protein from N. punctiforme. The Ns-HO1 protein polypeptide chains can form eight main α- helices, in which the two α- helices at the end of the N and C terminus together with the fourth helix provided the bottom support for the advanced structure, and other helices extend and fill the bottom surface to form a sandwich-like structure of the Ns-HO1 molecule. In its advanced structure, the helixes (first, fifth and seventh helix) formed by several peptide segments containing the amino acids of the heme prebinding site is located on the periphery of the Ns-HO1 molecule; by side view, these branch helices provide important conditions for the formation of Ns-HO1 sandwich space for substrate heme anchoring and timely releasing of catalyzed products. This study provides a basis for further understanding and application of the biological functions and resources of heme oxygenase gene Ns-HO1 of N. sphaeroides.