Tuberculosis (TB), one of the deadliest infectious diseases, is caused by Mycobacterium tuberculosis (MTB) and remains a public health problem nowadays. Conventional MTB DNA detection methods require sophisticated infrastructure and well-trained personnel, which leads to increasing complexity and high cost for diagnostics and limits their wide accessibility in low-resource settings. To address these issues, we have developed a low-cost photothermal biosensing method for the quantitative genetic detection of pathogens such as MTB DNA on a paper hybrid device using a thermometer. First, DNA capture probes were simply immobilized on paper through a one-step surface modification process. After DNA sandwich hybridization, oligonucleotide-functionalized gold nanoparticles (AuNPs) were introduced on paper and then catalyzed the oxidation reaction of 3,3',5,5'-tetramethylbenzidine (TMB). The produced oxidized TMB, acting as a strong photothermal agent, was used for the photothermal biosensing of MTB DNA under 808 nm laser irradiation. Under optimal conditions, the on-chip quantitative detection of the target DNA was readily achieved using an inexpensive thermometer as a signal recorder. This method does not require any expensive analytical instrumentation but can achieve higher sensitivity and there are no color interference issues, compared to conventional colorimetric methods. The method was further validated by detecting genomic DNA with high specificity. To the best of our knowledge, this is the first photothermal biosensing strategy for quantitative nucleic acid analysis on microfluidics using a thermometer, which brings fresh inspirations on the development of simple, low-cost, and miniaturized photothermal diagnostic platforms for quantitative detection of a variety of diseases at the point of care.