With use of surface-enhanced Raman scattering (SERS) substrate, we are able to identify alkaloids of tobacco leaves. First, porous Si arrays were developed using metal-assisted chemical etching and followed by gold (Au) thin layer (50 nm) coating. Au decorated porous Si arrays provide a strong localized electric field and responsible for the enhancement of weak Raman signals of target molecules. Detection of model molecule rhodamine B at low concentration (10−9 M) confirmed the SERS activity of the developed substrates. The enhancement factor was calculated to be approximate 106. Further, developed substrates were utilized to identify alkaloid in solution of dry tobacco leaves (1 g and 0.25 g) and water (100 Ml). Raman peak 1030 cm−1 corresponding to standard nicotine was observed. Understanding of tobacco composition and their Raman vibration mode could be beneficial to decide the biomarker of various diseases related to tobacco chewing and smoking. This work highlights the level free detection of tobacco alkaloids and confirms that Raman spectroscopy technique can utilize for quality control, forensic and bioanalytical analyses of tobacco alkaloids at trace level. © 2019, Springer Nature Switzerland AG.