A device and method for the constant pressure regulation of microdroplet PCR in microfluidic chips are developed to optimize for the microdroplet movement, fragmentation, and bubble generation in microfluidic chips. In the developed device, an air source device is adopted to regulate the pressure in the chip, such that microdroplet generation and PCR amplification without bubbles can be achieved. In 3 min, the sample in 20 μL will be distributed into nearly 50,000 water-in-oil droplets exhibiting a diameter of about 87 μm, and the microdroplet will be subjected to a close arrangement in the chip without air bubbles. The device and chip are adopted to quantitatively detect human genes. As indicated by the experimental results, a good linear relationship exists between the detection signal and DNA concentration ranging from 101to 105copies/μL (R2= 0.999). The microdroplet PCR devices based on constant pressure regulation chips exhibit a wide variety of advantages (e.g., achieving high pollution resistance, microdroplet fragmentation and integration avoidance, reducing human interference, and standardizing results). Thus, microdroplet PCR devices based on constant pressure regulation chips have promising applications for nucleic acid quantification.