The invention relates to a method for preparing thymidine by a gradient pH method. The method comprises the steps of mixing DNA hemicellulosic hydrolyzate as a substrate with thymine, preparing the thymidine by using immobilizedEscherichia coli cells as a catalyst, adding a phosphoric acid buffer solution by using the gradient pH method in the preparation process to adjustthe pH value of the system in the early stage and the late stage of the reaction,effectively increasing the reaction rate and greatly shortening the reaction time, preparing immobilized Escherichia coliby an embedding-cross-linking method, and improving the resistance of bacteria to the substrate; added activated carbon can reduce the internal diffusion resistance of the immobilized Escherichia coli, facilitate the diffusion of substances in the immobilized Escherichiacoli, and effectively increase the reaction rate,the immobilized Escherichia coli cells have good operational stability, and the reusability is high. The method for preparing the thymidine is simple in process, the conversion rate is good, the yield is over 61%, and the purity of the thymidine after purification is over 96%.