The invention discloses a segmented connection structure of a prefabricated concrete culvert and a construction method of the segmented connection structure. The segmented connection structure of the prefabricated concrete culvert comprises an upper culvert section and a lower culvert section. The upper culvert section is of a frame type structure with a downward opening. The lower culvert section is of a frame type structure with an upward opening and corresponds to the frame type structure, with the downward opening, of the upper culvert section. Connecting keys are symmetrically arranged on the two side edges of the frame type structure of the upper culvert section. Embedded square grooves are formed in the positions, corresponding to the connecting keys, on the two side edges of the frame type structure of the lower culvert section. The connecting keys are arranged in the embedded square grooves to fix the upper culvert section and the lower culvert section. The connecting keys are arranged on the upper culvert section, the embedded square grooves are formed in the lower culvert section, the connecting keys are placed in the embedded square grooves, and therefore connection of the upper culvert section and the lower culvert section is achieved, and an integral structure is formed. Field pouring is not needed, machining only needs to be conducted in a factory, the field construction time is shortened, field construction equipment is omitted, the cost is reduced, and the field operation time is shortened while the construction quality is guaranteed.