Nanostructures have been multiplying the advantages of Raman spectroscopy and further amplify the advantages of Raman spectroscopy is a continuous effort focused on the appropriate design of nanostructures. Herein, we designed different shapes of plasmonic nanostructures such as Vertical, Zig Zag, Slant nanorods and Spherical nanoparticles employing the DC magnetron sputtering system as SERS-active substrates for ultrasensitive detection of target molecules. The fabricated plasmonic nanostructures sensitivity and uniformity were exploited by reference dye analyte. These nanostructures were utilized in the label free detection of infectious disease, Tuberculosis (TB). For the first time, TB detection from serum samples using SERS has been demonstrated. Various multivariate statistical methods such as principal component analysis, support vector machine, decision tree and random forest were developed and tested their ability to discriminate the healthy and active TB samples. The results demonstrate the performance of the SERS spectra, chemometric methods and potential of the method in clinical diagnosis.