A single-tube on-beam quartz enhanced photoacoustic spectroscopy (SO-QEPAS) spectrophone, which employs a custom-made quartz tuning fork (QTF) having a prong spacing of 700 μm and operating at the 1st overtone flexural mode, is reported. The design of QTF prong geometry allows the bare QTF to possess twice higher Q-factor values for the 1st overtone resonance mode falling at ∼17.7 kHz than in the fundamental resonance mode at ∼2.8 kHz, resulting in an 8 times higher QEPAS signal amplitude when operating in the 1st overtone resonance mode. Both the vertical position and length of the single-tube acoustic micro-resonator (AmR) were optimized to attain optimal spectrophone performance. Benefiting from the high overtone resonance frequency and the quasi 1st harmonic acoustic standing waves generated in the SO-QEPAS configuration, the AmR length is reduced to 14.5 mm. This allows the realization of compact spectrophone and facilitates the laser beam alignment through the QTF + AmR system. The signal enhancement in the overtone resonance mode and the high acoustic coupling efficiency between the AmR and QTF in the SO-QEPAS configuration yields an overall sensitivity enhancement factor of ∼380 with respect to the bare custom QTF operating in the fundamental resonance mode.