Pistacia chinensis seed oil is proposed as a promising non-edible feedstock for biodiesel production. Different extraction methods were tested and compared to obtain crude oil from the seed of Pistacia chinensis, along with various deacidification measures of refined oil. The biodiesel was produced through catalysis of sodium hydroxide (NaOH) and potassium hydroxide (KOH). The results showed that the acid value of Pistacia chinensis oil was successfully reduced to 0.23 mg KOH/g when it was extracted using ethanol. Consequently, the biodiesel product gave a high yield beyond 96.0%. The transesteriﬁcation catalysed by KOH was also more complete. Fourier transform infrared (FTIR) spectroscopy was used to monitor the transesterification reaction. Analyses by gas chromatography-mass spectrometry (GC-MS) and gas chromatography with a flame ionisation detector (GC-FID) certified that the Pistacia chinensis biodiesel mainly consisted of C18 fatty acid methyl esters (81.07%) with a high percentage of methyl oleate. Furthermore, the measured fuel properties of the biodiesel met the required standards for fuel use. In conclusion, the Pistacia chinensis biodiesel is a qualified and feasible substitute for fossil diesel.