Laminar burning velocity is a crucial characterization parameter in fuel combustion progress. Investigating the combustion properties is significant to improve combustion efficiency. This paper employs a constant volume combustion bomb and high-speed ripple shadow camera technology to explore the influence of equivalence ratios (0.8–1.4) and ammonia energy shares (0%–40%) on the laminar burning velocity of n-dodecane/ammonia mixture at an ambient temperature of 400 K and a pressure of 1 bar. The results illuminated that the unstretched laminar burning velocity of n-dodecane increased from 46 cm/s at ϕ = 0.8 to 61 cm/s at ϕ = 1 and then decreased to 32 cm/s at ϕ = 1.4. When the ammonia energy ratio reaches 40%, the maximum laminar burning velocity experiences a reduction of 44% because the blended ammonia inhibited the formation of H and O radicals based on kinetic analysis in the fast reaction zone. With the increase of ammonia ratio, the Markstein length of mixed burned gas gradually increases. Particularly under a 1.4 equivalent ratio condition, the Markstein length increases from 0.2 mm to 0.8 mm, indicating that ammonia components significantly enhance flame stability. To gain a deeper understanding of the influence mechanisms of laminar burning velocity, a simplified Yao-Otomo (Y-O) model with 92 species and 355 reactions is proposed, coupling the mechanisms of n-dodecane and ammonia. The Y-O model has demonstrated a predictive trend that is consistent with more detailed WUT-NH3 model (176 species and 2839 reactions), and the Y-O model reduces the amount of computation to save calculation time, which has a higher engineering applicability. The turbulent kinetic energy (TKE) induced by premixed fuel in engine cylinders is estimated through comprehensively analyzing the laminar burning velocity and flame thickness characteristics. The findings indicate a 45% reduction in the peak value of TKE as the ammonia energy ratio increases.