The PCR technique is indispensable in biology and medicine, but some difficulties are associated with its use, including false positive or false negative amplifications. To avoid these issues, a non-amplification nucleic acid detection protocol is needed. In the present study, we propose a method in which nucleic-acid probe hybridization is combined with thio-NAD cycling to detect nucleic acids without amplification. We report our application of this method for the detection of the gene of MPT64 in Mycobacterium tuberculosis. Two different cDNA probes targeted the mpt64 gene: the first probe was used to immobilize the mpt64 gene, and the second probe, linked with alkaline phosphatase (ALP), was hybridized to a target sequence in the mpt64 gene. A substrate was then hydrolyzed by ALP, and a cycling reaction was conducted by a dehydrogenase with its co-factors (thio-NAD and NADH). The single-stranded DNA, double-stranded DNA, plasmid DNA for the mpt64 gene, and whole genome of M. tuberculosis var. BCG were detected at the level of 105-106 copies/assay, whereas the non-tuberculosis mycobacteria (e.g., M. avium, M. intracellulare, M. kansasii, and M. abscessus) were below the limits of detection. The present method enables us to avoid the errors inherent in nucleic acid amplification methods.