Two new half-sandwich cyclopentadienyl ruthenium(II) complexes containing α-amino acids, [CpRu(PPh3)2(Ser)] (Ser = l-serine) and [CpRu(PPh3)(Met)] (Met = l-methionine), were synthesized and characterized by physicochemical methods. Interactions of these two complexes with calf thymus DNA were investigated by UV–Vis absorption spectroscopy, emission spectroscopy and competitive binding studies. The results indicate that both complexes can interact with DNA, leading to the damage of the double helix. [CpRu(PPh3)2(Ser)] binds to DNA by intercalation, while the binding mode for [CpRu(PPh3)(Met)] is more complicated due to the formation of an EB-DNA-complex (EB = ethidium bromide). The affinity of the Met complex for DNA is stronger than that of the Ser complex, which could be due to groove–surface combination or electrostatic interaction in addition to intercalative binding.