Assays using probes labeled with electrochemiluminescent moieties are extremely powerful analytical tools that are used in fields such as medical diagnostics, environmental analysis and food safety monitoring, in which sensitive, reliable and reproducible detection of biomolecules is a requirement. The most efficient electrochemiluminescence (ECL) reaction to date is based on tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)3(2+)) with tripropylamine (TPrA) as the co-reactant. Here we present a detailed protocol for preparing Ru(bpy)3(2+) probes and their bioanalytical applications. This protocol includes (i) the synthesis of a biologically active Ru(bpy)3(2+)-N-hydroxysuccinimide (NHS) ester, (ii) its covalent labeling with both antibodies and DNA probes and (iii) the detection and quantification of ECL in a microfluidic system with a paramagnetic microbead solid support. In our magnetic bead-based ECL system, two probes are required: a capture probe (labeled with biotin to be captured by a streptavidin-coated magnetic bead) and a detector probe (labeled with Ru(bpy)3(2+)). The complex consisting of the analyte, the capture probe, the detector probe and the magnetic bead is brought into contact with the electrode by using a magnetic field. The Ru(bpy)3(2+) reacts with TPrA in solution to generate the ECL signal. The full protocol, including the synthesis and labeling of the bioactive Ru(bpy)3(2+), requires 5-6 d to complete. ECL immunoassays or nucleic acid tests only require 1.5-2 h, including the sample preparation time.