A novel fluorescent, hydrophobic cellulose-based UV-blocking film was developed via chemical surface modification. Cellulose acetoacetate (CAA) film was firstly regenerated by acetyl acetylation of cellulose in a homogeneous system. Based on this esterified film with acetoacetyl groups, both 1,4-dihydropyridine (DHP) fluorescent ring and hydrophobic octadecylamine (ODA) were bonded to cellulose chains through Hantzsch reaction and the formation of enamine bond, by using a one-pot process under room temperature. The resulting film exhibited bright fluorescent and excellent UV-blocking properties (shielding almost 100% UV), which are very stable even being exposed to long periods of UV radiation, harsh temperature treatment and different solvents. In addition, this film has good hydrophobic feature (the contact angle reach up to 112°). This work provided a facile and environmentally friendly strategy to fabricate multifunctional cellulose-based film, which had great application potential in the field of UV protection, fluorescent materials, packaging and cover materials for greenhouse.