The invention discloses a homogeneous method for preparing a wood fiber derivative under room temperature. The method comprises the following steps: drying ground wood fiber powder, suspending the dried wood fiber powder in a solvent, and stirring till the wood fiber is dissolved completely to obtain wood fiber solution; dissolving an acylation reagent in the same solvent, and stirring under the room temperature till the acylation reagent is dissolved completely to obtain acylation reagent solution; adding the acylation reagent solution into the wood fiber solution, and carrying out esterification reaction under the room temperature and a moisture isolated condition; and depositing the solution obtained after the esterification reaction, collecting, washing, freezing and drying deposits, and preparing to obtain the required wood fiber derivative. The invention has the advantages that homogeneous derivatization of wood fiber is realized under the room temperature; and not only can the homogeneity and the stability of the product be improved effectively, but also equipment requirements and energy requirements can be reduced. Therefore, the invention has a significant scientific value and practical significance on the development and the utilization of renewable resources such as wood fiber, biological substances and the like.