Based on the combination of tunable diode mid-infrared laser absorption spectroscopy technology and long optical path multiple-reflection technology, the fast and real-time detection of trace acetylene in nmol/mol level is studied by using C2H2 gas absorption spectrum near 3025.7 nm in mid-infrared region. The driving current of the interband cascade laser is modulated by the superimposition of the high frequency sine wave generated by DDS AD9958 and triangle wave signal, and then the stable drivingof laser is realized through constant current circuit. HgCdTe photodetector is used to receive mid-infrared laser, and target signal is extracted by integrated lock-in amplifier. A long optical path absorption cell is designed based on the combination ofWhite multiple-reflection and plane reflection, and its measured optical path reaches 19.2 m, which further reduces the detection limit of C2H2. In laboratory, trace C2H2 gas sample is measured with the concentration ranging from 0 to 1000 nmol/mol. It shows that the linear error is less than ±1% F.S., and the detection limit is 0.29 nmol/mol, which indicates that the method has the advantages of high accuracy, no cross interference from background gas and convenient use.