A stepwise chemical reaction strategy based on the specific recognition of boronic acid to diol, and N-hydroxysuccinimide (NHS) ester to amine group, was designed to construct a "signal on" electrochemiluminescence (ECL) platform for highly sensitive detection of dopamine. A boronic acid-functionalized pyrene probe was synthesized and was self-assembled on the sidewalls of carbon nanotubes via π-π stacking interactions as capture probes on a glassy carbon electrode. Meanwhile, 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) (DSP)-functionalized CdTe quantum dots (QDs) were designed as signal probes and characterized with transmission electron microscopy and spectroscopic techniques. Upon stepwise chemical reaction of dopamine with boronic acid and then DSP-QDs, the QDs were captured on the electrode as ECL emitters for signal readout, leading to an ultralow background signal. By using O2 as an endogenous coreactant, the "signal on" ECL method was employed to quantify the concentration of dopamine from 50 pM to 10 nM with a detection limit of 26 pM. Moreover, the stepwise chemical reaction-based biosensor showed high specificity against cerebral interference and was successfully applied in the detection of dopamine in cerebrospinal fluid samples. The stepwise chemical reaction strategy should be a new concept for the design of highly selective analytical methods for the detection of small biomolecules.