A novel and high-performance biosensing platform was prepared on the basis of one-pot biochemical preoxidation and electropolymerization of monomer (BPEM) for high-load and high-activity immobilization of enzymes. As representative materials here, 2,5-dimercapto-1,3,4-thiadiazole (DMcT) was used as the monomer, glucose oxidase (GOx) was used as the model enzyme, and enzymatically generated H2O2 (EG-H2O2) in the presence of glucose was used as the preoxidant. In the BPEM protocol, glucose was added to a pH 7.0 phosphate buffer suspension containing ultrasonically dispersed DMcT and GOx, in order to enzymatically generate H2O2, which preoxidized DMcT to DMcT oligomers/polymer (DMcTO) and thus led to the formation of DMcTO-GOx composites with a great deal of GOx entrapped; the composites were then co-electrodeposited with poly(DMcT) on a Au electrode. For comparison, the enzyme immobilization was also conducted by a preoxidation-free conventional electropolymerization protocol (CEP), as well as a chemical preoxidation and electropolymerization of monomer (CPEM) protocol with externally added H2O2 (EA-H2O2) as the preoxidant. The glucose biosensors constructed by the BPEM and CPEM protocols exhibited detection sensitivities enhanced by 119 and 88 times, respectively, compared to that constructed by the CEP protocol, as well as limits of detection lowered by ca. 2 orders of magnitude. The higher sensitivity of the enzyme electrode prepared by the BPEM protocol compared to that prepared by the CPEM protocol is probably due to the improved proximity of biochemical preoxidation around the enzyme molecules and thus a larger enzyme load. The electrochemical quartz crystal microbalance technique was used to investigate various electrode modification processes, which also revealed that poly(DMcT) could be cathodically detached from the electrode surface, favorably enabling the electrochemical regeneration of the electrode substrate. The proposed BPEM strategy is recommended for wide biosensing and biocatalysis applications based on many other polymers/enzymes.