Urban green spaces (UGSs) are ecological systems that provide the connection between cities and nature. UGSs offer several ecosystem services (ESs) not only in relation to their aesthetic and recreational values but also for their effects on life’s quality, health and wellbeing. These abilities are essential to respond to the challenge of global climate change, but the effectiveness of ESs provided by UGSs depends on their health status. Chlorophyll fluorescence (ChlF) is one of the main methodological approaches to study “urban plant physiology”. Here, a review of the studies on the methodological application of ChlF in urban environment is reported and detailed in order to evaluate the potential of this technique in assessing the performance of urban vegetation and its ESs. The reviewed literature (1994-2015) concerning the use of ChlF for the detection of photosynthetic efficiency in UGSs reveals that this technique is (i) increasingly used in researches performed in urban environments; (ii) suitable for several experimental conditions; (iii) suitable for every types of vegetation, allowing the screening of species and genotypes with tolerance to the urban environment; (iv) a powerful tool to asses urban plant stress conditions in vivo; (v) based on several methods and instruments usable in urban environments.