Abstract To examine the methodology for determining the moisture diffusion behavior of lignocellulosic biomass in steady and unsteady states (two stages of a sorption isotherm), the diffusion coefficients in the steady and unsteady states (D _SS and D _US) were investigated over a range of relative humidity (RH) from 10 to 90% using a dynamic vapor sorption (DVS) apparatus and a specifically designed cell kit. Thin samples with a thickness of 50 μm were prepared from three lignocellulosic biomasses, i.e. poplar, Chinese fir and moso bamboo. Based on Fick’s first and second laws, D _SS and D _US were determined. An increase in D _SS or D _US was observed with increasing equilibrium moisture content (EMC) or transient status, regardless of the lignocellulosic biomass species. The moisture-dependent D _SS of poplar, Chinese fir and moso bamboo was similar to values previously reported. Chinese fir and moso bamboo exhibited the highest and the lowest D _SS values, respectively, when the same EMCs were achieved. The results of this study revealed that D _SS and D _US of lignocellulosic biomass (even with limited dimensions) could be determined during a sorption isotherm in a wide humidity range. Furthermore, the results are helpful for simulating moisture transport behaviors in the fields of drying, paper packaging and wooden building maintenance.