In this study, a novel catalyst based on layered double hydroxides (LDHs) attached by hexamethylene-1,6-diisocyanate (HMDI) and citric acid (LDHs-g-HMDI-Citric acid) is reported and used to increase the yield of biurets synthesis. Biuret derivatives 5a-n were prepared by reaction of several phenyl allophanates (3a-d), which prepared from the reaction of phenyl chloroformate and urea derivatives (2ad), with variously substituted amines (4a-g) in the presence of LDHs-g-HMDI-Citric acid as a reusable heterogeneous catalyst at reflux condition for 60-180 min. These biurets (5a-n) were evaluated for human immunodeficiency virus type-1 (HIV-1) protease inhibitory activity by HIV-1 p24 antigen ELISA kit and six of them (5n, 5i, 5j, 5 m, 5f, and 5a) showed moderate activity on HIV-1 virus with IC 50 values ranging from 55 to 100 μM compared with the azidothymidine as the reference drug (IC 50 = 0.11 μM). Results of the in vitro test and docking study were in good correlation.