<p>Nanocatalysts, including nanozymes, photocatalysts and sonocatalysts, have been investigated to trigger catalytic reactions in vivo to regulate biological microenvironments and stimulate therapeutic effects. Compared with lower metal atom utilization rate and catalytic activity of conventional nanocatalysts, single-metal atom catalysts (SACs) usually possess higher catalytic activity and selectivity owing to their well-defined structures and maximized atom utilization. Their properties are, however, strongly dependent on their composition and the preparation procedure. Here we describe the design, preparation and functionalization of SACs with single-metal atoms positioned within nitrogen-doped carbon supports. The SACs are prepared by pyrolysis of zeolitic imidazolate framework-8 (ZIF-8) or polydopamine-derived materials. Their properties depend on, for example, the metal chosen and atoms available for coordination; four example procedures are described: Cu–N₄ from Cu–ZIF-8, Ir–N₅ from Ir@ZIF-8 plus melamine, Co–PN₃ from triphenylphosphine@Co-ZIF-8 and Cu–SN₃ from ZnS@Cu-polydopamine. These SACs need to be functionalized to, for example, reduce aggregation and in vivo corona formation before they can be used in biological applications. In this Protocol, functionalization with the proteins (that is, cholesterol oxidase and pyruvate oxidase) is used as an example. The Protocol provides advice regarding physicochemical and functional characterization, as well as for performing experiments in tumor-bearing mice. The functional experiments were designed with the aim of identifying nanocatalysts with peroxidase-like activity that generate reactive oxygen species within areas of the tumor microenvironment that have increased levels of hydrogen peroxide. SAC synthesis takes 3–4 days, functional modification requires one extra day and the most basic and essential in vitro and in vivo assays require 2–3 months.</p>