Wood structures have been recently considered as one of the important building styles due to their green use of natural resource, low energy consumption, and less waste emission. But wood structures are vulnerable to near surface material deterioration over long duration, as the result of mechanical impact, chemical corrosion and fungi attack. In order to maintain such natural structure in designed mechanical performance, it is necessary to detect the near surface wood defect with non-destructive evaluation (NDE) methods on a regular basis. Among the existing NDE methods, acoustic-laser technique has been demonstrated for detecting near surface detect in a variety of structures. When using this technique in wood structure detection, the measurement accuracy may be affected by the wood grain. Taking into account this issue, the present research work aims to understand the wood grain on the dynamic behavior of near surface layer in wood structure under acoustic excitation and assess the grain effect on the defect detection via acoustic-laser technique. In this study, artificial defects are fabricated in two wood beams, with the transverse and parallel directions to the grain respectively. Vibration characteristics measured by acoustic-laser technique for the two grain cases are compared and discussed. This paper also provides the suggestions of practical application of the technique in detecting wood structure in considering the grain effect.