DNA immobilization at specific locations on the inner wall of microchannels could lead to many interesting applications. In this paper, three procedures (electrostatic adsorption, thiol-Au covalent bonding, and electrochemical entrapment) were carried out for the immobilization of DNA probes onto stainless steel microdot electrodes inside a polydimethylsiloxane (PDMS) microchannel. The microdot electrodes were fabricated via crossed microwire moulding, a modified soft lithographic technique, to be naked on the inner wall of the PDMS microchannel. The immobilization of DNA probes onto the surface of the microdot electrodes was performed by microfluidics. It was found that, for DNA electrostatic adsorption, different electrode behaviours were exhibited inside and outside the microchannel; in the microchannel, the negatively charged DNA probes would undergo abnormal electrostatic adsorption onto the cathode. Furthermore, DNA immobilization through thiol-Au covalent bonding and DNA entrapment in enhanced polypyrrole (PPy)-modified dots inside microchannels was demonstrated. Further work is needed, but present results are promising and suggest a route by which microfluidics may be married with label-free electrochemical detection and with very low-cost DNA diagnostics, which could prove invaluable.