Cholesterol (CHO) in human blood is one of the most frequently and crucially quantified substances in diagnostic laboratories. However, the visual and portable point of care testing (POCT) methods have been rarely developed for the bioassay of CHO in blood samples. Here, we developed the electrophoresis titration (ET) model, chip device of ~60 grams, and quantification method for the POCT of CHO in blood serum based on moving reaction boundary (MRB). In this model, the selective enzymatic reaction is integrated with ET Chip for visual and portable quantification. At first, CHO reacted with cholesterol oxidase (CHOx) in the anode, producing H2O2 and Cholest-4-en-3-one in the solution. H2O2 further oxidized the colorless and chargeless Leucocrystal Violet (LCV) dye in to the violet positive charge crystal Violet (CV+) and, under the influence of the electric field, the CV+ migrates in the ET channels and were titrated by the alkali of sodium hydroxide immobilized in the ET channels. The length covered by MRB measured was the function of the CHO content. The relevant experiments validated the feasibility of the model and method. Further, the experiments revealed the high selectivity, portability, and visuality of the ET-MRB model, device, and method. Finally, the experiments showed a fair sensitivity of LOD of 5 μM, good linearity of 10-1000 μM (r2 = 0.9919), the fair stability (intra-day RSD of less than 5.09%, inter-day RSD of less than 6.36%), and high recovery (99.4%-105%). All the data and results indicating the potential of the ET-MRB model, chip device, and method for POCT of CHO in human blood samples.