Bioluminescent bacterial biosensors can be used in a rapid in vitro assay to predict sensitivity to commonly used chemotherapy drugs in acute myeloid leukemia (AML). The nucleoside analog cytarabine (ara-C) is the key agent for treating AML; however, up to 30 % of patients fail to respond to treatment. Screening of patient blood samples to determine drug response before commencement of treatment is needed. To achieve this aim, a self-bioluminescent reporter strain of Escherichia coli has been constructed and evaluated for use as an ara-C biosensor and an in vitro assay has been designed to predict ara-C response in clinical samples. Transposition mutagenesis was used to create a cytidine deaminase (cdd)-deficient mutant of E. coli MG1655 that responded to ara-C. The strain was transformed with the luxCDABE operon and used as a whole-cell biosensor for development an 8-h assay to determine ara-C uptake and phosphorylation by leukemic cells. Intracellular concentrations of 0.025 μmol/L phosphorylated ara-C were detected by significantly increased light output (P < 0.05) from the bacterial biosensor. Results using AML cell lines with known response to ara-C showed close correlation between the 8-h assay and a 3-day cytotoxicity test for ara-C cell killing. In retrospective tests with 24 clinical samples of bone marrow or peripheral blood, the biosensor-based assay predicted leukemic cell response to ara-C within 8 h. The biosensor-based assay may offer a predictor for evaluating the sensitivity of leukemic cells to ara-C before patients undergo chemotherapy and allow customized treatment of drug-sensitive patients with reduced ara-C dose levels. The 8-h assay monitors intracellular ara-CTP (cytosine arabinoside triphosphate) levels and, if fully validated, may be suitable for use in clinical settings.