An Au‐on‐Au tip sensor is developed for the detection of
Salmonella typhimurium( Salmonella), using a new synthetic nucleic acid probe (NAP) as a linker for the immobilization of a DNA‐conjugated Au nanoparticle (AuNP) onto a DNA‐attached thin Au layer inside a pipette tip. In the presence of Salmonella, RNase H2 from Salmonella(STH2) cleaves the NAP and the freed DNA‐conjugated AuNP can be visually detected by a paper strip. This portable biosensor does not require any electronic, electrochemical or optical equipment. It delivers a detection limit of 3.2×103 CFU mL−1 for Salmonellain 1 h without cell‐culturing or signal amplification and does not show cross‐reactivity with several control bacteria. Further, the sensor reliably detects Salmonellaspiked in food samples, such as ground beef and chicken, milk, and eggs. The sensor can be reused and is stable at ambient temperature, showing its potential as a point‐of‐need device for the prevention of food poisoning by Salmonella.