In this Letter, a differential integrating sphere-based photoacoustic spectroscopy (PAS) gas sensor is proposed for the first time to our knowledge. The differential integrating sphere system consists of two integrating spheres and a tube. Based on differential characteristics, the photoacoustic signal of the designed differential integrating sphere was doubly enhanced and the noise was suppressed. Compared with a single channel integrating sphere, the differential integrating sphere sensing system had a 1.86 times improvement in signal level. An erbium-doped fiber amplifier (EDFA) was adopted to amplify the output of diode laser to enhance the optical excitation. The second harmonic (2f) signal of differential integrating sphere-based acetylene (C2H2) PAS sensor with an amplified 1000 mW optical output power was 104.67 mV, which was 22.80 times improved compared to the sensing system without EDFA. When the integration time was 100 s, the minimum detection limit (MDL) of the differential integrating sphere-based C2H2 PAS sensor was 416.7 ppb. The differential integrating sphere provides a new method, to the best of our knowledge, for the development of PAS sensor, which has the advantages of photoacoustic signal enhancement, strong noise immunity, and no need for optical adjustment.