This report describes the fabrication of a novel microfluidics nanobiochip based on a composite comprising of nickel oxide nanoparticles (nNiO) and multiwalled carbon nanotubes (MWCNTs), as well as the chip's use in a biomedical application. This nanocomposite was integrated with polydimethylsiloxane (PDMS) microchannels, which were constructed using the photolithographic technique. A structural and morphological characterization of the fabricated microfluidics chip, which was functionalized with a bienzyme containing cholesterol oxidase (ChOx) and cholesterol esterase (ChEt), was accomplished using X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy. The XPS studies revealed that 9.3% of the carboxyl (COOH) groups present in the nNiO-MWCNT composite are used to form amide bonds with the NH2 groups of the bienzyme. The response studies on this nanobiochip reveal good reproducibility and selectivity, and a high sensitivity of 2.2 mA/mM/cm2. This integrated microfluidics biochip provides a promising low-cost platform for the rapid detection of biomolecules using minute samples.