Silver nanoparticles (Ag NPs) are among the most widely produced and used nanomaterial due to their antimicrobial and antibacterial properties, allowing a wide range of commercial applications. Thereby, the increasing use of Ag NPs should inevitably lead to the release and accumulation of these NPs into the environment, resulting in adverse effects on plants, animals and humans. Chlorophyll fluorescence (ChlF) has been proposed as a non-destructive and accurate tool for detecting the impacts of environmental stress on plants. Little is known about the photophysical behavior of plants when exposed to a metallic NPs-containing environment. The present study evaluated the interaction between chlorophyll (Chl) and Ag NPs, over a wide range of nanoparticle concentrations (from 0 μM to 200.0 μM), by monitoring the ChlF. The results reveal that the ChlF is quenched in the presence of Ag NPs, as a result of the static and dynamic quenching processes. The present results suggest that ChlF has a great potential to be used in the future as an analytical tool for monitoring the interaction of plants and NPs as well as investigating the effects of NPs on plants.