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In this study, we propose an interference-free SERS-based aptasensor for trace detection of chlorpyrifos (CPF) in real samples. In the aptasensor, gold nanoparticles coated with Prussian blue (Au@PB NPs) were employed as SERS tags to provide a sole and intense Raman emission at 2160 cm−1, which could avoid overlapping with the Raman spectrum of the real samples in 600–1800 cm−1 to improve the anti-matrix effect ability of the aptasensor. Under the optimum conditions, this aptasensor displayed a linear response for CPF detection in the range of 0.1–316 ng mL−1 with a low detection limit of 0.066 ng mL−1. In addition, the prepared aptasensor shows excellent application to determine CPF in cucumber, pear and river water samples. The recovery rates were highly correlated with high-performance liquid chromatography‒mass spectrometry (HPLC‒MS/MS). This aptasensor shows interference-free, specific and sensitive detection for CPF and offers an effective strategy for other pesticide residue detection. Interference-free SERS-based Aptasensor for Chlorpyrifos Detection. [Display omitted] • An interference-free SERS aptsensor for chlorpyrifos residue detection was firstly developed. • PB containing CN was used as Raman report with a sole Raman emission in Raman-silent region (1800-2800 cm−1). • The aptsensor showed a linear detection range of 0.1–316 ng mL−1 with a LOD of 0.031 ng mL−1. • The aptsensor was successfully appiled to detect chlorpyrifos in real samples. [ABSTRACT FROM AUTHOR]