Typically dealing with practical samples with very complex matrices, ambient ionization mass spectrometry suffers from low detection sensitivity. In this study, molecular imprinting technology was explored and integrated with the membrane electrospray ionization (MESI) method for direct sample analyses. By enriching targeted analytes on molecularly imprinted membranes (MIMs), improvement (by 10- to 50-fold) in the limit of quantitation could be achieved, compared to conventional nanoelectrospray ionization methods or other ambient ionization methods. MIMs were prepared by cross-linking a synthesized molecularly imprinted polymer layer onto a polyvinylidene difluoride (PVDF) membrane. The characteristics of MIM in recognizing target analytes were investigated and verified. Experiments showed that MIM-ESI could provide satisfactory performances for direct quantification of targeted analytes in complex samples using mass spectroscopy (MS), and the quantitative performance of this methodology was validated. With the capability of target enrichment, the uses of MIM-ESI MS in different application fields were also demonstrated, including food safety, quantification of drug concentrations in blood, pesticide residues in soil, and antibiotic residues in milk.