Gold nanoparticles (AuNPs) are known for various biosensing applications owing to their unique physicochemical properties, ease of synthesis, surface functionalization, tunable optical properties with size and shape, etc. These unique properties of AuNPs allow sensing and detecting the presence of chemical compounds/biological molecules, aiding in their application in different fields of biomedical engineering. The principles of various biosensors like colorimetric biosensor, chemiluminescence biosensor, optical biosensor, electrochemical biosensor, and piezoelectric biosensor are discussed in detail in this chapter. The multiple biorecognition elements like enzymes and antibodies are also discussed. The different strategies and novel approaches for the design and development of biosensors including deoxyribonucleic acid (DNA)–functionalized AuNP-based colorimetric assay, dual gold nanostructure–based electrochemical immunosensor, interdigitated device electrode–based biosensor, and microfluidics-based biosensor are discussed with their respective applications. The different examples showing enhanced sensing properties attributed to functionalization with AuNPs have also been included in the chapter. The applications of AuNPs for detection of cancer and infectious diseases are also discussed in detail in this chapter. Fluorescence and electrochemiluminescence of AuNP-based systems for DNA detection along with detection of small biomolecules and metabolites like cholesterol, hematin, uric acid, and proteins using AuNPs for the diagnosis of venous thromboembolism, Parkinson's disease, etc., are explored in this chapter. In addition to the application of AuNPs for detection of various analytes and metabolites of biological relevance, we have also explored the application of AuNPs for agricultural and environmental applications. The possible and probable future applications of AuNPs and the advent of wearable sensors are also discussed with multiple examples. © 2023 Elsevier Ltd. All rights reserved.