Electrochemical techniques were used for estimating xylazine HCl (XLZ) in bulk powder, medicinal manufacturing and human serum. Electro-oxidation of XLZ at carbon multiwalled nanotube (MWCNT), 1-n-butyl-3-methylpyridinium hexafluorophosphate ion crystal (BMH) and sodium dodecyl sulfate (SDS) MWCNT-BMH-SDS electrode in 0.04 M Britton–Robinson buffer (BR) with pH 7.0, was studied in numerous buffer structures and at different pH values. The experimentation and instrumental parameters to assessable commitment of XLZ had been optimized, and a detection limit was observed as 4.80 nM. The precision and accuracy for the recognized method was tested by retrieval studies with good repeatability and reproducibility of the estimated method. The projected method was practiced successfully to the dosage form and spiked serum.