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Mapping of the metabolic activity of tumor tissues represents a fundamental approach to better identify the tumor type, elucidate metastatic mechanisms and support the development of targeted cancer therapies. The spatially resolved quantification of Warburg effect key metabolites, such as glucose and lactate, is essential. Miniaturized electrochemical biosensors scanned over cancer cells and tumor tissue to visualize the metabolic characteristics of a tumor is attractive but very challenging due to the limited oxygen availability in the hypoxic environments of tumors that impedes the reliable applicability of glucose oxidase-based glucose micro-biosensors. Herein, the development and application of a new glucose micro-biosensor is presented that can be reliably operated under hypoxic conditions. The micro-biosensor is fabricated in a one-step synthesis by entrapping during the electrochemically driven growth of a polymeric matrix on a platinum microelectrode glucose oxidase and a catalytically active Prussian blue type aggregate and mediator. The as-obtained functionalization improves significantly the sensitivity of the developed micro-biosensor for glucose detection under hypoxic conditions compared to normoxic conditions. By using the micro-biosensor as non-invasive sensing probe in Scanning Electrochemical Microscopy (SECM), the glucose uptake by a breast metastatic adenocarcinoma cell line, with an epithelial morphology, is measured., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)