A new electrochemical assay performed directly on fresh tumor biopsies is described. Substrate mediated electrochemical biosensor was used to screen biopsy slices for colon cancer via electrochemical determination of enzymatic activity to distinguish between cancerous and normal epithelial tissues. The screen was based on the downregulation of the enzyme alkaline phosphatase (ALP) in intestine epithelial cancerous cells. The ALP activity recorded from fresh biopsy samples removed from colon tumors induced in mice was compared to that from samples collected simultaneously from the small and large healthy intestines of same mice. Samples were suspended in a specially designed multichamber electrochemical sensor. Upon the addition of the substrate p-aminophenol phosphate yielding p-aminophenol resulting from the enzymatic reaction, a voltage was applied between a working and a reference Ag/AgCl microelectrode and current was recorded. Significant differences in current signal were observed for biopsies removed from tumors compared with samples removed from healthy tissue. Reproducibility was demonstrated. The new technology allows a direct assay carried out on small volume slices from fresh biopsies without the need for pretreatment and labor intensive preparative steps. The new assay offers rapid and straightforward detection providing results obtained within minutes of biopsy removal.