The dual direct injection strategy can flexibly control the reactivity distributions and equivalence ratio, which shows a great potential in realizing clean and efficient combustion. However, the sprays’ collision is inevitable when the fuel sprays are injected into the combustion chamber simultaneously owing to limited in-cylinder space, which is rarely investigated. The investigation aims to explore the atomization and combustion characteristics of collision between biodiesel and butanol sprays at different injection delay times of biodiesel (0, 0.5, 1, 1.5, 2 ms). Experiments are conducted in a constant volume combustion chamber. The collision spray and combustion images are captured by optical diagnosis techniques. Several macroscopic parameters are obtained, including spray behavior, spray area, ignition delay, ignition location, flame behavior, flame lift-off length, and natural luminosity intensity. Results show that the variation of injection delay time can effectively change the spray distribution and realize the concentration stratification. The liquid-phase diffusion region increases with an increased premixed charge of butanol spray, but the total evaporation time has a tiny difference. The sprays’ collision can promote the ignition process, but the ignition location is slightly affected by the injection delay time of biodiesel. A trade-off relationship between the vapor-phase diffusion area and combustion characteristics is found. When the injection delay time exceeds 0.5 ms, a longer injection delay time can lead to a larger vapor-phase area, which causes smaller equivalent ratio, longer flame-off length, and lower luminosity intensity. However, a longer injection delay would lead to an uncomplete combustion of the n-butanol spray. At the experimental cases, the injection delay time of biodiesel should be 1/2–2/3 injection duration to realize complete combustion and small emissions.