Aggregation behavior of nanoparticles in water, particularly in the presence of surface coating, is a fundamental behavior to understand as it closely relates to the material function, reactivity, and toxicity. Engineered DNA, an important type of surface coating, is essentially different from conventional polymeric coatings with homogeneous repeating units. The heterogeneous nature of DNA with numerous combinations of the four nucleotides poses a challenge to understand the aggregation of nanoparticles coated with them. In this study, we use both experimental and computational approaches to study how DNA properties influence the aggregation of DNA-conjugated gold nanoparticles (DNA-AuNPs). Aggregation kinetics under different pH values and in three salts (NaCl, CaCl 2 , and MgCl 2 ) were investigated. Overall, DNA-AuNPs demonstrated similar aggregation behavior of electrosterically stabilized nanoparticles, where the surface coating layer thickness played a determining role. The underlying interactions between DNA-AuNPs were analyzed by an extended DLVO model, and the excluded volume repulsion, which varied as a function of layer thickness, was the dominant stabilizing repulsion. Interestingly, the DNA sequence was found to cause different layer thicknesses and thus profoundly different aggregation behaviors in the presence of cations. The interactions between DNA strands with different sequences and surrounding counterions were investigated via molecular dynamics simulations. The simulation results were consistent with experimental observations and supported the sequence-dependent conformational change of DNA coating. Our study provides new molecular-level insights into aggregation of nanoparticles with heterogeneous surface coating such as DNA.