In the world, environmental pollution has increased due to industrialization. To reduce environmental pollution, the use of hemp, which has a low environmental impact and is known for its carbon-negative feature, has gained importance in the production of composites. The aim of this research is to develop a bio-composite lightweight building material using expanded perlite (EP) and hemp stalks. In this study, 11 different mixtures were produced by using polyester matrix as binder, hemp and EP as filling material. These produced bio-composites' physical, mechanical, and thermal conductivity were examined. The results show that while the increase in the amount of filling in the composite, there are decreases in apparent density, compressive strength, ultrasonic pulse velocity (UPV) and thermal conductivity. However, there was an increase in water absorption, swelling in thickness (ST) and weight loss (WL) values. The water absorption, UPV, and compressive strengths of composites were increased, while the thermal conductivity was decreased, by the addition of EP. The lowest thermal conductivity value was reached as 0.1093 W/mK in the H35P5-coded specimen. In addition, the highest compressive strength value was reached as 35.175 MPa in the H25P5-coded specimen, except for the reference specimen. According to the thermogravimetric analysis results the addition of EP and hemp caused the thermal stability of the composite to deteriorate at lower temperatures. The results showed that sustainable biocomposites from EP and hemp stalks can be used as thermal insulation materials. Also the use of carbon-negative hemp with a low environmental impact makes the study important.