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In this paper, the naphthalimide fluorescence probe M1 with Fe3+ fluorescence system was successfully constructed, and its UV–visible absorption spectrum and fluorescence emission spectrum were investigated. Under optimal experimental conditions, the system has good anti-interference ability. The M1 probe has good linearity with Fe3+ concentration (1 × 10−6–15 × 10−6 mol·L−1). The detection limit reaches 7.55 × 10−8 mol·L−1. The complexation ratio of M1 probe to Fe3+ is 1:1. For the first time, we used the constructed M1 probe with Fe3+ fluorescence system to determine Fe3+ concentration in the Chinese herbal medicines, Panacis Quinquefolii Radix and Paeoniae Radix Alba, and in the fluorescence imaging of HeLa cells.