The interaction of DNA with different block copolymers, namely poly (trimethylammonium chloride methacryloyoxy)ethyl)-block-poly(acrylamide), i.e., (PTEA)-b-(PAm), and poly (trimethylammonium chloride methacryloyoxy)ethyl)-block-poly(ethylene oxide), i.e., (PTEA)-b-(PEO), was studied. The nature of the cationic block was maintained fixed (PTEA), whereas the neutral blocks contained varying amounts of acrylamide or (ethylene oxide) units. According to results from isothermal titration microcalorimetry measurements, the copolymers interaction with DNA is endothermic with an enthalpy around 4.0 kJ mol−1 of charges for (PTEA)-b-(PAm) and 5.5 kJ mol−1 of charges for (PTEA)-b-(PEO). The hydrodynamic diameters of (PTEA)-b-(PEO)/DNA and (PTEA)-b-(PAm)/DNA polyplexes prepared by titration were around 200 nm at charge ratio (Z+/−) < 1. At Z+/− close and above 1, the (PTEA)50-b-(PAm)50/DNA and (PTEA)50-b-(PAm)200/DNA polyplexes precipitated. Interestingly, (PTEA)50-b-(PAm)1000/DNA polyplexes remained with a size of around 300 nm even after charge neutralization, probably due to the size of the neutral block. Conversely, for (PTEA)96-b-(PEO)100/DNA polyplexes, the size distribution was broad, indicating a more heterogeneous system. Polyplexes were also prepared by direct mixture at Z+/− of 2.0, and they displayed diameters around 120–150 nm, remaining stable for more than 10 days. Direct and reverse titration experiments showed that the order of addition affects both the size and charge of the resulting polyplexes.