A series of amine-based dendrimer polyamidoamine (PAMAM) demulsifiers with different initial cores were synthesized and investigated in the demulsification process of diesel-in-water emulsions. With the aim of systemic evaluation of their demulsification performance, some important factors of the demulsification processes were investigated including demulsifier dosage, settling time, temperature, oil content, and kinds of diesel. The demulsifier with the triethylenetetramine (TETA) initial core provided excellent demulsification performance by removing oil with less dosage and at relatively low temperature in short periods and reached 96.66% demulsification efficiency for catalytic cracking diesel emulsion. The results showed its good application prospects. In order to gain insight into the demulsification process and mechanism, some measurement methods were adopted. Micrograph and droplet size distribution of emulsions illustrated that the PAMAM demulsifier could lead to the breakup of diesel-in-water emulsions by flocculation and coalescence. The surface tension and interfacial tension gave a basic understanding of the demulsification mechanism. Zeta potential indicated that emulsion had been broken up. The conductivity measurement explained the demulsification mechanism from the aspect of the electrostatic interactions of moving droplets. The dendrimer and SDS had strong aggregation interactions in the system according to the results of hydrodynamic radium.