In the present work, three-dimensional porous HxTiS2 nanosheet-polyaniline (PANI) nanocomposites were first synthesized by a two-step method. First, HxTiS2 ultrathin nanosheets were prepared by the lithium intercalation and exfoliation method, followed by the surface polymerization reactions of aniline. The influences of the amount of HxTiS2 nanosheets on the nanocomposite morphology and electrochemical performances of the nanocomposites modified glass carbon electrode (HxTiS2 nanosheet-PANI/GCE) were investigated. The results demonstrated that the incorporation of HxTiS2 nanosheets as a suitable substrate can regulate the growth of PANI, enhance the electrode stability and improve interfacial electron transfer rates. In addition, based on the nanocomposites, we developed a novel electrochemical sensor to directly detect trace Cu(2+), and discovered that the coordination interaction between Cu(2+) cations and the N atoms of the imine moieties in PANI endowed the electrochemical sensor with high selectivity. Because of the synergetic effects of HxTiS2 nanosheets and PANI, the as-prepared electrochemical sensor exhibited highly sensitive and selective assaying of Cu(2+) with a detection limit of 0.7 nM (signal-to-noise ratio (S/N) equal to 3) and a linear range from 25 nM to 5 μM, under optimal conditions.