The vials easily crack in the filling process of freeze-dried products. Cracks and loose assembly of the vial plugs causes air to enter the vials, and thus leads to the metamorphism or degeneration of the freeze-dried products. A measurement system based on tunable diode laser absorption spectroscopy (TDLAS) was developed for fast and non-invasive leakage detection of vials. By analyzing the mole fraction difference of water vapor between the detection light path and the reference light path (Δx) and mole fraction of water vapor (xr) of the reference light path, the threshold at different conditions is obtained. The measurement accuracy of the system is validated firstly, and it reveals that the standard deviation of the system is less than 0.007%. Then, ten vials are detected when xr is 1.29%, 1.33%, 1.89%, 2.20%, respectively. Vials No.4 and No.8 are considered as leaked vials, since Δx is larger than the corresponding threshold at various mole fractions of water vapor xr, and the results are in good agreement with the real situation.