The threat of food safety and the limited analytical methods with high performance promote the growing interest in the development of pathogenic bacteria biosensors. This study presents a pathogenic bacteria biosensing system, where a novel three-dimensional (3D) chip acts as an analytical carrier and DNA-programmed hybridization chain reaction (HCR) causes signal amplification. The 3D chip is designed featuring a compact multichannel structure. It has a large surface area for sensitive sensing and exhibits multiple functions of target capture, separation, rinsing, and signal detection to simplify the analysis processes. HCR, which enables the fluorophore's polymerization, is designed as two signal amplification modes, each with unique advantages. Mode I achieves highly sensitive detection in a "sandwich" assay format, in which a long HCR-amplified probe is used to boost the fluorescence signal. In mode II, the assembly of HCR is performed on the inner surface of the 3D chip. Especially, a group of rapid-assembly HCR sequences is proposed, of which the assembly time as short as 15 min stands out among the related works previously reported. Under the optimal conditions, the proposed biosensing system has the limits of detection (LOD) of 4 and 8 cfu/mL in mode I for Staphylococcus aureus detection and in mode II for Salmonella enterica Typhimurium detection, respectively. The specificity and the real sample applications are evaluated. This multichannel-structured 3D chip based on HCR signal amplification has potential applications in food safety monitoring and biosensor development.