Protein kinase C (PKC), a protein which is widely present in organisms, plays a pivotal role in the pathogenesis of many diseases, and has become a target for the diagnosis and treatment of many illness. In this study, a rapid method based on liquid crystal (LC) biosensor and nano-modified signal amplification technology for PKC detection is demonstrated. Glutaraldehyde (GA) was used to fix the PKC antibody/gold nanoparticles (anti-PKC/AuNPs) conjugations to the substrate mixed self-assembled with (3-aminopropyl)-triethoxysilane/N, N-dimethyl-N-octadecyl-(3-aminopropyl)-trimethoxysilyl chloride (APTES/DMOAP). The above processing maintained the LC molecules to arrange vertically and induced a dark background under polarized light. When PKC was presented, it would be fixed on substrate through binding with anti-PKC of the complex specifically. The specific binding event resulted in the change in morphology of the substrate, arrangement of the LC molecules and brightness of the LC film, thereby the sensitive detection of PKC was achieved. The developed biosensor present a good linear relationship between the average gray intensity of the optical imaging and the PKC concentration in a wide linear range of 10–250 ng·mL−1, the correlation coefficient was 0.997, and has a very low detection limit of 1.680 ± 0.005 ng·mL−1. The findings of this work provided a potential application for the high sensitivity and simple detection of protein kinases.