In this study, a novel sample pretreatment strategy of solid-phase microextraction using graphene oxide molecularly imprinted polymers as adsorbents coupled with high-performance liquid chromatography was developed to detect norfloxacin in the marine environment. As a carrier, the imprinted polymers were synthesized by precipitation polymerization with graphene oxide. Compared with graphene oxide non-imprinted polymers, the graphene oxide molecularly imprinted polymers exhibited higher adsorption capacity towards norfloxacin. The synthesized polymeric materials were packed into a molecularly imprinted solid-phase microextraction cartridge, and critical parameters affecting the extraction process were optimized. Under the optimized molecular imprinted solid-phase microextraction condition, the proposed method was applied to the analysis of norfloxacin for seawater and fish with satisfactory recovery (90.1–102.7%) and low relative standard deviation (2.06–5.29%, n = 3). The limit of detection was 0.15 μg L⁻¹ and 0.10 μg kg⁻¹ for seawater and fish, respectively. The study revealed that the proposed molecularly imprinted solid-phase microextraction represents an attractive sample pretreatment strategy for the analysis of norfloxacin in the marine environment.