Application-level middleware in Express is a function that runs for every request to the app or specific routes. When a request comes in, Express runs middleware functions in the order they were added. Each middleware receives the request and response objects, plus a next function. Calling next() passes control to the next middleware or route handler. If next() is not called, the request stops and no further processing happens. After middleware, the route handler runs and sends the response. This flow ensures middleware can modify requests or responses before the final handler sends data back to the client.