Cellular and brain metabolism of dopamine can be correlated with a number of neurodegenerative disorders, and as such, in vivo analysis of dopamine in the presence of structurally related neurotransmitters (NT) represents a holy grail of neuroscience. Interference from those NTs generally does not allow selective electroanalysis of dopamine, which redox transformation overlaps with those of other catecholamines. In our previous work, we reported an electrochemical RNA-aptamer-based biosensor for specific analysis of dopamine (Analytical Chemistry, 2013; Vol. 85, p 121). However, the overall design of the biosensor restricted its stability and impeded its operation in serum. Here, we show that specific biorecognition and electroanalysis of dopamine in serum can be performed by the RNA aptamer tethered to cysteamine-modified gold electrodes via the alkanethiol linker. The stabilized dopamine aptasensor allowed continuous 20 h amperometric analysis of dopamine in 10% serum within the physiologically important 0.11 μM range and in the presence of catechol and such dopamine precursors and metabolites as norepinephrine and L-DOPA. In a flowinjection mode, the aptasensor response to dopamine was ∼1 s, the sensitivity of analysis, optimized by adjusting the aptamer surface coverage, was 67 ± 1 nA μM−1 cm−2, and the dopamine LOD was 62 nM. The proposed design of the aptasensor, exploiting both the aptamer alkanethiol tethering to the electrode and screening of the catecholamine-aptamer electrostatic interactions, allows direct monitoring of dopamine levels in biological fluids in the presence of competitive NT and thus may be further applicable in biomedical research.