Engineered short-chain single stranded DNA (ssDNA) are emerging materials with various environmental applications, such as aptasensor, selective adsorbent, and hydrological tracer. However, the lack of fundamental understanding on the interactions of such materials with natural organic matter (NOM) hinders the improvement of their application performance in terms of sensitivity, selectivity, and stability. In this study, we investigated the interactions of ssDNA (four strands with systematically varied length and sequence) with two humic acids (Suwannee River humic acid (SRHA) and Aldrich humic acid (AHA)) and two humic-like NOM present in local aquatic matrices (ROM in river water and WOM in wastewater). Detailed, molecular level interaction mechanisms were obtained by probing the colloidal stability of the ssDNA-coated gold nanoparticles, coupled with product characterization using a suite of microscopic and spectroscopic techniques. Our study revealed that pi-pi interactions and divalent cation bridging were the major mechanisms for ssDNA-NOM interactions. ssDNA preferentially interacted with NOM with high aromaticity (AHA > SRHA/WOM/ROM). With divalent cations present (especially Ca2+), even a small amount of AHA could completely shield ssDNA, whereas the extent of shielding by SRHA/WOM/ROM depended on the relative content of ssDNA and NOM and whether bridges formed. The extent of shielding of ssDNA by NOM provides a potential answer to the reported conflicting effects of natural water matrices on the performance of DNA-based sensors. Taken together, our findings provide insights into the transformations of engineered ssDNA under environmentally relevant conditions as well as implications for their performance optimization in practical aquatic applications (e.g., from DNA design to pretreatment strategy).