Antibiotic resistance threatens human health worldwide. Patients infected with antibiotic-resistant bacteria require appropriate antibiotic prescriptions based on a rapid antibiotic susceptibility test (AST). Various rapid AST methods have been developed to replace the conventional AST method, which requires a long testing time. However, in most cases, these methods require a high density of bacterial samples, which leads to an additional incubation or concentration process. In this study, we introduce a rapid AST platform that allows the use of low-density bacterial samples by concentrating bacterial cells and performing AST on a single microfluidic chip. In addition, the outlet-free loading process enables the platform to load the sample and concentrate bacteria into a small field of view for single-cell detection. Using this method, rapid AST determined antibiotic resistance in three hours from a standard strain of 103 colony-forming unit (CFU) per ml bacterial concentration. This technique can be used for the cell-based drug testing of various low-concentration bacterial samples.