This research aims to improve the output power of self-pumping glucose enzymatic biofuel cell (EBFC) and modifying the anode. Adding a fixed ratio of methyl red-chitosan (MR-CS) can effectively improve the EBFC efficiency and stability. In addition, chitosan can be obtained from discarded crustacean fishery waste objects such as shrimp and oysters, are also significant to the use of environmentally friendly materials. The catalyst was immobilized on pyrenecarboxaldehyde (PCA), polyethyleneimine (PEI) and multi-wall carbon nanotubes (MWCNT) and combined with glucose oxidase (GOx). Finally, the [PCA/GOx]/PEI/Nafion solution/MWCNT/[MR-CS] catalyst was immobilized on the carbon cloth. Experimental analysis was progressed under the preparation of enzyme-supported electrode to observe the feasibility of the anode electrode. Experiment including Fourier transform infrared spectroscopy (FTIR) to analyze the distribution of functional groups after modification of the carbon cloth electrode, and through the comparison of the ultraviolet–visible spectrometer (UV–Vis), it can be known that the concentration ratio of [MR-CS] is 1:5, the glucose oxidase load can be maximized. Electrochemical analysis (Cyclic Voltammetry, CV) measures the activity of the maximum reaction of the anode material and the corresponding redox peak, and scanning electron microscope (SEM) observes the surface morphology of the modified electrode. Self-pumping glucose enzymatic biofuel cell module was assembled and examined, the results showed that the maximum output power density (MPD) was 2.64 mW/cm2.