Chitosan-based CNT composite fibers have received much attention due to their biocompatibility and biodegradability. However, further development and practical application of the fibers has been blocked due to their relatively low mechanical strength and electrical conductivity. In order to overcome these disadvantages and to extend the practical applicability of the composite fibers based on chitosan, a novel composite fiber based chitosan/MWNT composite and cross-linked polyurethane (PU) with 4,4′-methylenebis(phenyl isocyanate) (MDI) were newly developed herein using a wet-spinning technique. The experimental results confirmed that the proposed method was highly effective, allowing practical formation of the structures with enhancement of the mechanical strength and electrical conductivity by cross-linked PU as well as optimized MWNTs in the chitosan/MWNT composites fibers. Furthermore, the fabricated composite fibers with good mechanical strength (88.2 MPa) and excellent electrical conductivity (7.3 S cm−1) can be fluidly spun on any substrate.