n-Butanol, as a biological alternative fuel containing oxygen, has similar physical and chemical properties to gasoline and has a wide range of sources, which has attracted more and more attention and research. Direct injection technology has been widely used in the field of internal combustion engine due to its advantages of flexibility and control ability. In this paper, the secondary injection of n-butanol engine under the mode of in-cylinder direct injection is discussed to organize stratified combustion of the mixture, optimize combustion to improve the thermal efficiency, and reduce emission. A four-cylinder four-stroke spark ignition (SI) engine was selected to carry out the secondary injection experiment of n-butanol under the excess air ratio (λ) of 1, an engine speed of 1500 r/min, and a low load, and the variables were the second injection ratio and timing. The results show that the secondary injection of n-butanol can achieve stratified combustion of the mixture, but only at a specific second injection timing such as 100°CA before compression top dead center (BTDC) or 125°CA BTDC, the combustion effect is the best. A small second injection ratio can optimize combustion, improve brake thermal efficiency, and reduce hydrocarbon and carbon monoxide emissions. When the second injection ratio is greater than 60%, it results in incomplete fuel combustion, a 3 to 4% reduction in thermal efficiency, and an increase in emissions. Coefficient of variation (COV) was increased by secondary injection, but the effect was insignificant in the small injection ratio, and it will increase with the increase of the second injection ratio. The change of particle number is mainly affected by the nuclear particle number, and with the increase of the second injection ratio, the total particulate number is more affected by the second injection timing. The second injection ratio of 40% can reduce the total particle number under the mixed-gas stratification condition.