A label-free electrochemical immunosensor was developed for the ultra-sensitive detection of β-lactoglobulin (β-LG). The novel nanocomplex of carbon nanochips, colloidal gold nanoparticles and titanium dioxide nanoparticles TiO2/CNC/AuC were constructed on conducting polymer, chitosan, and were characterised using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). This nanocomplex interface was studied using cyclic voltammetry (CV) and showed great improvement at the gold electrode surface with enhanced electrochemical performance, sensitivity and selectivity for β-lactoglobulin. Under optimal parameters, the square wave voltammetry (SWV) response curve was determined from 0.01 pg/mL to 500 pg/mL using [Fe(CN)6]3−/4−] redox probe. The calibration plot illustrates a linear relationship between log β-LG concentration and SWV current, with the limit of detection determined to be 0.01 pg/mL. This immunosensor displayed high sensitivity, selectivity, reproducibility and stability, and can be utilised for the detection of β-LG in real food samples.