Over the last years, polymers have gained great attention as substrate material, because of the possibility to produce low-cost sensors in a high-throughput manner or for rapid prototyping and the wide variety of polymeric materials available with different features (like transparency, flexibility, stretchability, etc.). For almost all biosensing applications, the interaction between biomolecules (for example, antibodies, proteins or enzymes) and the employed substrate surface is highly important. In order to realize an effective biomolecule immobilization on polymers, different surface activation techniques, including chemical and physical methods, exist. Among them, plasma treatment offers an easy, fast and effective activation of the surfaces by micro/nanotexturing and generating functional groups (including carboxylic acids, amines, esters, aldehydes or hydroxyl groups). Hence, here we present a systematic and comprehensive plasma activation study of various polymeric surfaces by optimizing different parameters, including power, time, substrate temperature and gas composition. Thereby, the highest immobilization efficiency along with a homogenous biomolecule distribution is achieved with a 5-min plasma treatment under a gas composition of 50% oxygen and nitrogen, at a power of 1000 W and a substrate temperature of 80 °C. These results are also confirmed by different surface characterization methods, including SEM, XPS and contact angle measurements.