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A room-temperature broadly tunable mid-infrared difference frequency laser source for highly sensitive trace gas detection has been developed recently in our laboratory. The mid-infrared laser system is based on quasi-phase-matched (QPM) difference frequency generation (DFG) in a multi-grating, temperature-controlled periodically poled LiNbO3 (PPLN) crystal and employs two near-infrared diode lasers as pump sources. The mid-infrared coherent radiation generated is tunable from 3.2 μm to 3.7 μm with an output power of about 100 μW. By changing one of the pump laser head with another wavelength range, we can readily obtain other needed mid-infrared wavelength range cover. The performance of the mid-infrared laser system and its application to highly sensitive spectroscopic detection of CH4, HCI, CH2O, and NO2 has been carried out. A multi-reflection White cell was used in the experiment gaining ppb-level sensitivity. The DFG laser system has the features of compact, room-temperature operation, narrow line-width, and broadly continuous tunable range for potential applications in industry and environmental monitoring.