The extraction method with organic solvent extraction to detect nitric oxide was improved, and the production of nitric oxide in mice myocardium in vitro was detected with this method. Using organic solvent (DETC)₂-Fe²⁺-NO complex was extracted from water phase into ester phase, and nitric oxide in sample of large volume can be detected by ESR at room temperature. The extracting ability of several organic solvents such as ethyl acetate, butyl acetate, glycerol triacetate, iso-amy lacetate, and n-butanol, was compared, and it was found that ethyl acetate was a good kind of organic solvent. There was a good linear relationship between the concentration of nitric oxide and ESR intensity within concentration of 20 μmol/L, and the detected limit was improved to below 200 nmol/L; (DETC)₂-Fe²⁺-NO complex is easy to decompose in light but it is very stable in dark at 0～4℃ which, there is only a little change after ten days.