In this paper, a differential quartz-enhanced photoacoustic spectroscopy (D-QEPAS) sensor is reported. The differential photoacoustic cell (PAC) was used to generate the photoacoustic effect. Two quartz tuning forks (QTFs) with a quality factor (Q) up to 10 000 were used as the acoustic wave transducers. The signal of D-QEPAS sensor was doubly enhanced by the differential characteristic of differential PAC and resonant response of QTF. The background noise was suppressed based on the differential principle. With the help of the finite element method, the acoustic field characteristics were simulated and calculated. Wavelength modulation spectroscopy technique and second harmonic (2f) detection technique were applied to detect photoacoustic signal. Trace acetylene (C2H2) gas detection was performed to verify the D-QEPAS sensor performance. The 2f signal amplitude of differential mode was 116.03 μV, which had a 1.65 times improvement compared with the 2f signal amplitudes of QTF1. When the integration time was 334 s, the minimum detection limit of D-QEPAS sensor was about 496.7 ppb. The reported D-QEPAS provides a development and idea for the widely reported QEPAS technique.