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In this paper, an optical waveguide evanescent field fluorescence microscopy is studied. Based on Maxwell's equation, a seven‐layer theoretical analysis model is developed for the evaluation of an optical waveguide excitation fluorescence microscopy. The optical waveguide excitation fluorescence microscopy structure is systematically and comprehensively analysed at the wavelengths of 488, 532 and 646 nm for fluorescent dyes. The analysis results provide some useful suggestions, which will be beneficial to the research of an optical waveguide evanescent field fluorescence microscopy. LAY DESCRIPTION: An optical waveguide evanescent field fluorescence microscopy is studied in this paper. Considering the existence of cells, a seven‐layer theoretical analysis model of the optical waveguide excitation fluorescence microscopy is established. Based on this model, the effect of different parameters on the efficiency of optical waveguide excitation fluorescence microscopy is analysed. [ABSTRACT FROM AUTHOR]