Alkaline phosphatase (ALP) is an important enzyme-based biomarker present in several bacterial species; however, it is currently undervalued as a strategy to detect pathogenic bacteria. Here, we explore our ALP-responsive colorimetric and fluorescent probe (TCF-ALP) for such applications. TCF-ALP displayed a colorimetric and fluorescence response towards Staphylococcus aureus (S. aureus), with a limit of detection of 3.7 × 106 CFU mL-1 after 24 h incubation. To our surprise, TCF-ALP proved selective towards Staphylococcus bacteria when compared with Enterococcus faecalis (E. faecalis), and Gram-negative P. aeruginosa and E. coli. Selectivity was also seen in clinically relevant S. aureus biofilms. Owing to the high prevalence and surface location of S. aureus in chronic wounds, TCF-ALP was subsequently encapsulated in polyvinyl alcohol (PVA)-based hydrogels as a proof-of-concept "smart" wound dressing. TCF-ALP hydrogels were capable of detecting S. aureus in planktonic and biofilm assays, and displayed a clear colour change from yellow to purple after 24 h incubation using ex vivo porcine skin models. Overall, TCF-ALP is a simple tool that requires no prior knowledge, training, or specialist equipment, and has the potential to overcome issues related to invasive swabbing and tissue biopsy methods. Thus, TCF-ALP could be used as a tool to monitor the early development of infection in a wound and allow for the rapid provision of appropriate treatment for Staphylococcal bacterial infections.