Throughout the years, reported intracellular H2O2 sensors just focused on unrelated measurements of intracellular H2O2 generated from the stimulus of Cd2+, ascorbic acid (AA) etc., leading to difficulty in data interpretation. Here, a novel reduced graphene oxide quantum dots (rGO QDs)/ZnO hybrid nanofibers-based electrochemical biosensor for the detection of intracellular H2O2 released from cancer and normal cells under the stimuli of the corresponding anticancer drugs permits a quantitative study of the interaction between the target drug compound and the cancer cell, which is suitable for candidate drug screening. Nylon 6/6 nanofibers are used as robust templates for the facile fabrication of novel rGO QDs/ZnO hybrid nanofibers via electrospinning followed by a step hydrothermal growth method. The as-made sensor was applied to determine H2O2 released from a prostate cancer cell (PC-3) versus a noncancerous cell (BPH-1) under the stimuli of the corresponding anticancer drugs (apigenin, antisense CK2αetc.). The amount of H2O2 released from the PC-3 cancer cell is about (320 ± 12) amol per cell and about (210 ± 6) amol per cell for the BPH-1 noncancerous cell under the stimuli of specific therapy drug antisense CK2α. These results demonstrate that the rGO QDs/ZnO hybrid nanofibers-based electrochemical biosensor can efficiently detect the distinct amounts of H2O2 released from cancer and noncancer cells.