Salmonella screening is a key to ensure food safety in poultry supply chains. Currently available Salmonella detection methods including culture, polymerase chain reaction and enzyme-linked immuno-sorbent assay could not achieve rapid, sensitive, and in-field detection. In this study, different strategies for separation and detection of Salmonella were proposed, compared, and improved based on our previous studies on immunomagnetic separation and impedance biosensor. First, the coaxial capillary for immunomagnetic separation of target bacteria was improved with less contamination, and 3 strategies based on the improved capillary and immunomagnetic nanoparticles were compared to separate the target bacteria from sample and form the magnetic bacteria. The experimental results showed that the strategy of capture in tube and separation in capillary was the most suitable with separation efficiency of approximately 88%. Then, the immune gold nanoparticles coated with urease were used to label the magnetic bacteria, resulting in the formation of enzymatic bacteria, which were injected into the capillary. After the urea was catalyzed by the urease on the enzymatic bacteria in the capillary, different electrodes were compared to measure the impedance of the catalysate and the screen-printed electrode with higher sensitivity and better stability was the most suitable. This impedance biosensor-based bacterial detection strategy was able to detect Salmonella as low as 102 CFU/mL in 2 h without complex operations. Compared to the gold standard culture method for practical screening of Salmonella in poultry supply chains, this proposed strategy had an accuracy of approximately 90% for 75 real poultry samples.