Trace disinfection by-products (DBPs) produced during the disinfection of drinking water are potentially carcinogenic, teratogenic and mutagenic, which has aroused much attention recently. In this study, a molecularly imprinted (MIP) solid -phase microextraction (SPME) fiber coating was prepared by an in-situ polymerization method using a dummy template molecule for the analysis of trace 2,6-dichloroindole-1,4-benzoquinone (2,6-DCBQ), a typical DBP. The characterization results suggested that this monolithic SPME fiber under the optimized conditions had the porous structure, large surface area and good thermal stability. Due to the strong structural recognition and molecular interaction between MIP SPME coating and target molecule, it showed good extraction selectivity and capacity to trace 2,6-DCBQ with an imprinting factor of 4.7. Then, coupling with high-performance liquid chromatography (HPLC)-ultraviolet (UV) detection, a sensitive analytical method for trace 2,6-DCBQ in water samples was successfully established with a detection limit down to 2.3 ng/mL. The recoveries of the proposed method were in range of 84.4–122% with the relative standard deviations of 1.0–13% (n = 3). The results showed that this MIP SPME-HPLC-UV method possessed high analytical selectivity and sensitivity for trace 2,6-DCBQ in water, which would benefit the improvement of the practicability of DBPs monitoring and detection methodology.