The natural manipulative experiment, by controlling influencing factors appropriately to study changes in the ecosystem under natural conditions, has become more and more popular with ecologists. This article, which focused on the experimental design methods of urban ecology (such as artificial gradients, natural gradients and urban construction experiments), environmental factors (such as air pollution, heat islands, nightlight and surface hardening) and the response of urban ecosystems (such as communities, green spaces, green roofs and watersheds), reviewed a great number of cases using natural manipulative experiments in urban ecology at home and abroad. It also summarized the characteristics of the natural manipulative experiment of urban ecology, pointed out that the main problems which should be considered in the urban ecology manipulative experiment, and proposed the main development direction in the future:(1) Manipulative experiments on the compound impact of multiple environmental factors; (2) Manipulative experiments on the response of the ecosystem structure and function; (3) Long-term near-natural manipulative experiments; (4) Large-scale network of manipulative experiments; (5) Manipulative experiments on the urban ecological restoration and construction. We hope that this review can play a role in advancing the experimental research of urban ecology in China.