Aptamer-based sensors (aptasensors) are emerging as powerful diagnostic tools for the sensitive and selective detection of disease biomarkers and pathogenic targets. Aptamers are synthetic nucleic acid ligands that offer high affinity, stability, and design flexibility, making them attractive alternatives to antibodies. Here, we report the development of electrochemical aptasensors incorporating surface-immobilised aptamers for the label-free detection of disease- and pathogen-associated targets in biologically relevant media. For extracellular vesicle detection, these platforms achieve sensitive, clinically relevant detection, with clear impedance changes observed upon target binding. In parallel, aptamers have been conjugated to fluorescent quantum dots to develop a FRET-based aptasensor for the detection of metallo-β-lactamase as a marker of antimicrobial resistance. Together, these results demonstrate the versatility of aptasensor platforms and highlight their potential for rapid, low-cost diagnostics with future translation into portable point-of-care devices.