The invention relates to a frame assembling method in a JESD204B protocol. The method comprises the following steps that: determining data bit width input into a transmitting terminal transport layer in advance; sampling the data bit width by a converter to obtain S pieces of sampling samples; adding a control bit and a padding bit into each sampling sample to form an integral character, wherein the control bit and the padding bit are positioned on the tail of the character; according to byte numbers in each frame, judging whether the character is split or not; and according to a judgment result, carrying out sorting processing on a character or byte corresponding to each sampling sample, and mapping a sorting processing result to a corresponding channel to form a frame in each channel. By use of the frame assembling method in the JESD204B protocol, the frame which conforms to the requirements of the JESD204B protocol can be assembled.