Realizing accurate pesticide multiresidue detection in a complex matrix is still a challenge for point-of-care sensing methods. Herein, we introduced background-free and multicolor aptasensors based on bioorthogonal surface-enhanced Raman scattering (SERS) tags and successfully applied them to analyze multiple pesticide residues. The excellent anti-interference and multiplex capability are due to the application of three bioorthogonal Raman reporters involving 4-ethenylbenzenamine (4-EBZM), Prussian blue (PB) and 2-amino-4-cyanopyridine (AMCP) with alkynyl and cyano groups, which demonstrated apparent Raman shift peaks at 1993 cm−1, 2160 cm−1, and 2264 cm−1 in the biologically Raman-silent region, respectively. Ultimately, a detection range of 1–50 nM for acetamiprid, atrazine and malathion was achieved with detection limits of 0.39, 0.57 and 0.16 nM, respectively. The developed aptasensors were successfully used to determine pesticide residues in real samples. These proposed multicolor aptasensors offer an effective strategy for pesticide multiresidue detection with advantages of anti-interference, high specificity and high sensitivity.