Abstract The study aimed to extend the applications of surface-enhanced Raman scattering (SERS) – recently one of the most promising and widely employed technique in food safety and quality monitoring – towards determination of two analytes in a single step for the first time. Thus, an ultra-sensitive and selective SERS based detection platform for the simultaneous determination of pesticides namely acetamiprid (AC) and 2,4-dichlorophenoxyacetic acid (2,4-D) in matcha tea has been developed. Owing to the potential interference of different nutrients and pigments in complicated matrices, the matcha tea samples with and without spiking AC and 2,4-D, were percolated through a solid phase extraction cartridge. The eluate was introduced to the SERS Au@Ag nanostructure substrate for monitoring SERS signal with subsequent chemometrics modeling. Under the optimized experimental conditions, a linear calibration exhibited at GA-PLS model with RPD values of 6.53 and 6.23, and Rp values 0.9943 and 0.9923, respectively for AC and 2,4-D. The method was validated in both mixed and single spiking conditions successfully in terms of student's t- and F test at 95% confidence with satisfactory recovery percentages. An RSD value < 4.85% indicates good reproducibility of the proposed method. Highlights • SERS active Au@Ag sensor to simultaneously detect AC and 2,4-D in tea. • DFT calculations to identify characteristic peaks for building GA-PLS model. • Solid phase extraction for sample pretreatment and masking matrix interference. • The LODs found were much lower than the current MRL set by the European Commission. • Satisfactory recoveries were obtained in terms of the student's t- and F test at 95% confidence. [ABSTRACT FROM AUTHOR]