This paper describes the fabrication of an imprinted fluorescent nanoprobe based on SiO2-coated NaYF4: Yb, Er upconversion nanoparticles (UCNP) encapsulated with a molecularly imprinted polymer (MIP) for determination of acetamiprid. The fluorescent MIP nanoprobe was prepared using UCNP as the material for fluorescence signal readout, acetamiprid as template molecule, methylacrylic acid (MAA) as functional monomer, and ethyleneglycol dimethacrylate (EGDMA) as cross-linking agent. The molecular imprinting layers were immobilized on the surface of the [email protected] by polymerization which occurred between the double bonds. [email protected] shows a high selectivity towards acetamiprid with an imprinting factor (IF) of 7.84. When [email protected] combines with acetamiprid, the fluorescence of the [email protected] can be quenched due to the photo-induced electron transfer. Under optimum conditions, the fluorescence method shows a good linear relationship between the decreased fluorescence intensity (with excitation/emission peaks at 980/542 nm) and the variation of acetamiprid in the concentration range 20 to 800 ng mL−1. The limit of detection (LOD) is 8.3 ng mL−1. This fluorescence method was also successfully applied to detect acetamiprid in apple and strawberry samples. The recoveries range from 89.6 to 97.9%, with relative standard deviations between 1.6 and 2.9% (n = 5). Graphical abstractA simple fluorescence nanoprobe which integrates upconversion nanoparticles (UCNPs) and molecular imprinting polymer (MIP) was developed for the determination of acetamiprid. The limit of the detection was determined as 8.3 ng mL−1. The selectivity was enhanced by molecular imprinting, and the sensitivity was improved by the high sensitivity of the fluorescence emitted from the UCNPs.