This paper reports a portable fluorescence resonance energy transfer (FRET) aptasensor for ampicillin (Amp) detection using upconversion particles (UCNPs) as energy donors and Au nanoparticles (AuNPs) as energy acceptors. The optimal parameters of the detection system were investigated. Under the optimal conditions, it had a good linear relationship between the fluorescence intensities and Amp concentrations, a high coefficient of determination (R2) of 0.9939, a wide detection range of 10-100 ng/mL, and a low limit of detection (LOD) of 3.9 ng/mL; meanwhile, the aptasensor had high selectivity for Amp against the interference of other antibiotics, and had good recovery and repeatability. Also, its detection performance had been successfully validated by milk samples. Therefore, the developed aptasensor based on FRET between UCNPs and AuNPs has a good prospect for Amp on-site detection in milk with a portable upconversion detection instrument.