Abstract: We describe the application of a fluorescently labeled water-soluble core-shell molecularly imprinted polymer (MIP) for fluorescence immunoassay (FIA) to detect trypsin. p-aminobenzamidine (PAB), a competitive inhibitor of trypsin, was immobilized in the wells of a microtiter plate enabling the capture of trypsin in an oriented position, thus maintaining its native conformation. Fluorescent MIP nanoparticles which bound selectively to trypsin were used for quantification. The MIP was prepared by a multi-step solid-phase synthesis approach on glass-beads functionalized with PAB, orientating all trypsin molecules in the same way. The core-MIP was first synthesized, using a thermoresponsive polymer based on N-isopropylacrylamide, so as to enable its facile liberation from the immobilized template by a simple temperature change. The shell, mainly composed of allylamine to introduce primary amino groups for post-conjugation of fluorescein isothiocyanate (FITC), was grafted in situ on the core-MIP, whose binding cavities were still bound and protected by the immobilized trypsin. The resulting core-shell MIP was endowed with a homogeneous population of high affinity binding sites, all having the same orientation. The MIP has no or little cross-reactivity with other serine proteases and unrelated proteins. Our MIP-based FIA system was successfully applied to detect low trypsin concentrations spiked into non-diluted human serum with a low limit of quantification of 50 pM, which indicates the significant potential of this assay for analytical and biomedical diagnosis applications