Improving the nutritional quality of plants has emerged from the fact that macro- and micro- nutrients are limited in various agricultural areas. The aim of our study was the biofortification of broccoli sprouts with selenium nanoparticles (NSePs) and evaluation of growth parameters, assimilator pigments content, total phenols, glucosinolates content along with antioxidant capacity, in order to boost value added output, such as improved nutrition and food functionality. NSePs were prepared by reduction of NaHSeO3 using glucose as reducing agent, and characterized from structural and morphological point of view. The growth of broccoli seedlings was dependent on NSePs concentration. The treatment with 10 and 50 ppm NSePs caused a slight increase in total biomass, by contrast with 100 ppm treatment. Chlorophyll content, total carotenoid and total phenols content was not affected by the treatment of broccoli sprouts with different concentrations of NSePs. Instead, the content of individual glucosinolates varied between the samples, depending on the levels of NSePs. The highest antioxidant capacity was obtained for 100 ppm NSePs concentration. The effective uptake of NSePs was further demonstrated by FTIR spectroscopy and Hyperspectral Microscopy. NSePs did not induce any toxicity on broccoli sprouts. Moreover, broccoli supply with NSePs may target higher nutritional impact and health benefits. ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 3, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. *********