PC software-based portable cyclic voltammetry (PCV) systems have the advantages of portability, high performance, and real-time detection. In this paper, the PCV system used cyclic voltammetry (CV) as the main detection and analysis method and contained the following components: a three-electrode unit, a portable potentiostat, and PC software. The PC software was used as the system control and display, and a dynamic peak position adjustment (DPPA) algorithm for E. coli measurements based on thick biofilm modification on electrodes was designed especially for this system to realize the real-time correspondence between the measured results and the modified electrodes. The performance test results obtained by setting different detection parameters in the PCV system were compared with those of commercial electrochemical workstations. The difference was less than 4.99%, with a relative standard deviation less than 0.20%. An electrochemical biosensor based on a Prussian blue-multiwalled carbon nanotube-gold nanoparticle composite was developed for E. coli detection. After constructing an antibody-BSA-E. coli electrode modification on the sensor, experimental data processed by the DPPA algorithm showed that the logarithm (lg DfE.coli) of the E. coli dilution factor and the peak current response had a linear relationship. The PCV system could quickly and accurately detect E. coli concentrations with dynamic adjustment algorithms for biofilm-modified electrodes. Furthermore, the system could detect the electrochemical activities of various high-sensitivity biomolecules, showing great detection potential for on-site monitoring and meeting the requirements of real-time and portable detection in various food safety fields.