Mortise and tenon are very important parts of gas turbine dealing operation safety. Additionally, the temperature distribution of the turbine blade and disk is affected by the heat transfer characteristics in its gap. Then, the S-shaped mortise and tenon gap were numerically studied under rotating conditions, and the flow and heat transfer characteristics were analyzed. First, the heat transfer coefficient (HTC) of the mortise and tenon surfaces was measured with thermochromic liquid crystal. Then, the numerical method was verified using the test results, and the grid independence analysis was conducted. Finally, the flow and heat transfer characteristics of the gap under static and rotating conditions were numerically studied, five different Reynolds numbers (Re = 15,000, 20,000, 25,000, 30,000, 35,000) and five gap widths (d = 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm) were conducted and analyzed in detail. The results show that, under the rotating condition, the pressure distribution in the gap is different from that of the static condition; the pressure increases along the radial direction due to the action of centrifugal force and reaches its maximum value at the corner of the “S” shaped structure. With the increase in Re, the heat transfer intensity of the gap increases gradually. Additionally, the heat transfer intensity of the gap increases with an increase in its width.