Weissella viridescens is a spoilage bacterium commonly found in low-temperature meat products. In this work, after fifteen rounds including three counter selection rounds of whole-cell systemic evolution of ligands by exponential enrichment (SELEX) in vitro, a novel aptamer L3 that can specifically recognize W. viridescens was obtained with a dissociation constant (K) value of 68.25 ± 5.32 nM. The sequence of aptamer L3 was optimized by truncation and a new aptamer sequence TL43 was obtained with a lower K value of 32.11 ± 3.01 nM. Finally, a simple and rapid fluorescence polarization (FP) platform was constructed to detect W. viridescens, in which FAM-labeled complementary sequence (FAM-cDNA) was employed to generate FP signal and streptavidin was used to amplify FP signal. In the presence of target bacteria, FP value decreased owning to the dissociation of FAM-cDNA from streptavidin/biotin-TL43/FAM-cDNA complex. Under optimal conditions, the concentration of W. viridescens and FP value displayed a good linear relationship with the detection range from 10 to 10 cfu/mL. Moreover, the designed detection system had a good recovery rate of 90.6%-107.7% in smoked ham samples compared with classical plate counting method, indicating the great potential of the selected and truncated aptamer in practical biosensing applications.