The affinity of polybetaines of pyridiniocarboxylate structure that differed by the number n of methylene groups in the betaine moieties (n = 1, 2,4, and 5) to the tagged poly(methacrylic acid) or DNA was estimated by fluorescence quenching techniques. The revealed irregular change of the affinity with increase in n and the pronounced pH-induced stabilization of the complexes in neutral media may be particularly beneficial in the development of (bio)separation techniques.