The isotope analysis of iron was performed using laser ablation molecular isotopic spectrometry (LAMIS) for the detection of radioactive hotspots in nuclear power plant (NPPs). Using an optical double-pulse configuration, the emission bands from FeO (D-5 Delta(i) -> X-5 Delta(i)) and FeO2 (l(5)B(1) -> l(5)B(2)) transitions were investigated and characterized. The isotopic shifts of (FeO)-Fe-54/(FeO)-Fe-56 and (FeO2)-Fe-54/(FeO2)-Fe-56 molecular emissions were identified at each rotational branch line. The quantitative analysis for isotopically enriched iron samples was performed by partial least squares regression. The cross-validation revealed superior results with root mean squares errors of prediction of about 0.07%. Subsequently, the detection of iron isotopes was carried out on the surface of structural materials of stainless steel-304, Inconel-690, and Portland cement to verify the feasibility for the application to NPPs. The surface density of Fe-54 particulates on each matrix was evaluated, and the results were comparatively analyzed according to the matrix characteristics. The spatial distribution of Fe-54 particulates on the stainless steel was identified by resolving the emission responses with focused spots. The present study not only provided characteristic optical information on the iron oxide emission, but also demonstrated the feasibility of LAMIS for the detection of iron isotopes in structural materials of NPPs.