A fluorescence sensor composed of alendronic acid (ADA) @upconversion nanoparticles (UCNPs) and mitoxantrone dihydrochloride (MTX) combined with polymer chain reaction (PCR) was developed for rapid, specific and ultrasensitive determination of Salmonella typhimurium. MTX, which has a wide absorption band at 500–700 nm, can completely overlap the upconversion fluorescence (UCF) emission spectrum, leading to the reduction of UCF intensity. By intercalating MTX into DNA structure, the absorption intensity of MTX is obviously weak. Thus, the concentration of target can be quantified by response change of UCF intensity based on inner filter (IFE) between UCNPs and MTX coupled with PCR. The sensitivity of sensor for detection of Salmonella typhimurium is improved due to IFE and PCR amplification process, with a detection limit of 11 CFU/mL. In addition, the UCNPs-MTX PCR fluorescence sensor has been applied to sensing Salmonella typhimurium in chicken samples with satisfied results. The research work revealed a rapid and sensitive analytical method for determination of Salmonella typhimurium and provided an insight for the construction of PCR fluorescence sensor.