Abstract Background A two-dose primary regimen of INO-4800 DNA vaccine demonstrated only modest immunogenicity in the previous phase 2 clinical trial. This booster study aimed to evaluate the immunogenicity and safety of a booster dose of INO-4800 in adults previously received two-dose regimen of INO-4800. Methods Healthy adults who received two doses of INO-4800 (1.0 mg or 2.0 mg) at least 12 months ago in a previous phase 2 trial were eligible for this booster study, conducted in Danyang, Jiangsu Province, China. Eligible participants were stratified by primary vaccination dose (1.0 mg or 2.0 mg) and age group (18–59 years or ≥ 60 years), and subsequently randomized in a 1:1 ratio to receive a third dose of INO-4800 or placebo at the same dosage as previously administered. The primary immunogenicity endpoint was the geometric mean concentrations (GMCs) of spike-binding antibodies on day 14 post-booster. The primary safety endpoint was the occurrence of adverse reactions within 14 days. Results Between December 20 and 23, 2021, 200 eligible participants were enrolled. 100 eligible participants who received two doses of 2.0 mg INO-4800 were randomly assigned (1:1) to receive a third dose of 2.0 mg INO-4800 (n = 50) or 2.0 mg placebo (n = 50). Another 100 eligible participants who received two doses of 1.0 mg INO-4800 were randomly assigned (1:1) to receive a third dose of 1.0 mg INO-4800 (n = 50) or 1.0 mg placebo (n = 50). On day 14 post-booster, the GMCs of spike-binding antibodies were significantly higher in 2.0 mg INO-4800 group ( 260.1 BAU/mL) compared to placebo group (2.8 BAU/mL, p