In recent years, significant progress has been achieved in the sensing and imaging of molecular oxygen (O(2)) in biological samples containing live cells and tissue. We review recent developments in the measurement of O(2) in such samples by optical means, particularly using the phosphorescence quenching technique. The main types of soluble O(2) sensors are assessed, including small molecule, supramolecular and particle-based structures used as extracellular or intracellular probes in conjunction with different detection modalities and measurement formats. For the different O(2) sensing systems, particular attention is paid to their merits and limitations, analytical performance, general convenience and applicability in specific biological applications. The latter include measurement of O(2) consumption rate, sample oxygenation, sensing of intracellular O(2), metabolic assessment of cells, and O(2) imaging of tissue, vasculature and individual cells. Altogether, this gives the potential user a comprehensive guide for the proper selection of the appropriate optical probe(s) and detection platform to suit their particular biological applications and measurement requirements.