Protein polymer conjugates have been developed in many fields. Most hybrids are composed of one protein attached to one or several polymer chains. The other form of hybrid involves the construction of multiple proteins on one polymer chain, thereby facilitating protein assemblies that provide multivalent effects. Unfortunately, synthetic methods for production of these types of hybrids are limited and challenging because precise control of the conjugation sites is needed. Herein, a novel synthetic polymer that can enzymatically assemble multiple proteins was developed. Polyacrylamide grafted with multiple microbial transglutaminase (MTG)-recognizable peptide derivatives was synthesized, and MTG-catalyzed site-specific conjugation of proteins with the polymer was achieved. The application for immunological biosensing was demonstrated using the assembly of a fusion protein composed of antibody-binding and enzyme moieties. This enzymatic method to synthesize a one-dimensional protein assembly on a synthetic polymer is versatile and can be expanded to a wide range of applications.