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High sensitivity is essential for the application of bioelectrochemical system-based sensor (BES sensor) in water quality early-warning, where the electroactive biofilm is of vital importance as it delivers a responsive electric signal to toxic substances. This study artificially designed the morphology of a naturally formed biofilm by employing a serrated knife to scrape the biofilm and thus obtained a reduced thickness and roughness. Then it was further cut by half to halve the biomass. BES sensors equipped with control and processed biofilms were operated under constant anode potential (CAP) and tested at different Cu(II) concentrations to study their sensitivities. Results revealed that the scraped biofilms delivered much increased sensitivity towards Cu(II) shock, which was attributed to a reduced thickness as illustrated by macroscopic and microscopic morphology analysis. Another finding was that biomass per unit interfacial area, rather than the biomass, also affected the sensitivity. To further describe how the inner biofilm responded the toxicity after morphology design, a one-dimension mass transfer model was developed to simulate the mass transfer of Cu(II) in the biofilms with different thicknesses. The relative threshold value of inlet Cu(II) concentration was employed to fit the modeling and experimental results, indicating that decreased biofilm thickness was beneficial for improving the sensitivity. Unlabelled Image • Biofilm's morphology design leads to reduced thickness and roughness. • Scraping the biofilm raises the sensitivity by 34%. • Biofilm's thickness is recognized essential factor on sensitivity. • Biomass per unit interfacial area affects the sensitivity. • One-dimension model simulates the Cu(II) transfer in biofilm. [ABSTRACT FROM AUTHOR]