A microfluidic chip integrated with a microheater and a luminescent temperature sensor for rapid, spatial melting curve analysis was developed and applied for the screening of a breast cancer gene fragment. The method could detect genetic differences in around 3 minutes total for the whole procedure, which is much faster than established procedures. A microfabrication technique was developed to allow for bonding of a temperature sensing thin film and a Pt microheater with PDMS and the chips could be employed to generate and measure thermal gradients and the fluorescence intensity of stained DNA through multispectral optical imaging. The sensing layer consisting of poly(styrene-co-acrylonitrile) and a tris(1,10-phenanthroline)ruthenium(II) temperature probe was generated by blade coating on a glass substrate with an attached Pt microheater. Calibration of the temperature between 20 and 90 °C yielded an overall resolution of around 0.13 K. The chip was employed for the screening of the BRCA 2 breast cancer gene; BRCA2 exon 5 was differentiated by its mutant rs80359463 by a 1.1 K difference in melting temperature and two fragments of BRCA2 exon 11 were differentiated by their mutants rs276174826 and rs876660311 by 0.7 K and 2.0 K, respectively. The standard deviations were between 0.1 and 0.5 K. Capable of detecting fluorescence in the DNA and temperature simultaneously and being imaged in a customized assembly, this microchip can be used to screen for mutations in a variety of DNA samples in disease diagnosis and prognosis.