Azithromycin (AZM) is a widely used macrolide antibiotic to treat infections caused by bacteria. Considering the side-effect to human health, a rapid and selective electrochemiluminescence (ECL) sensor was fabricated on the basis of molecularly imprinted polymer (MIP) modified carbon paste electrode as a recognition element. We systematically optimized the factors influencing the processes on the extraction and determination for AZM, including the amount of MIP power and paraffin oil, extraction solution, extraction time and the pH of Ru (bpy)(3)(2+) solution. Under the optimal condition, the wide linear range was observed from 1.0 x 10(-10) to 4.0 x 10(-7) mol L-1, and the limit of detection (LOD) was low as 2.3 x 10(-11) mol L-1 (S/N = 3). The spiked recoveries of AZM in urine and blood samples were from 98.4% to 113.5% with RSD changing from 3.7% to 13.2%, respectively. The results of AZM determination by this proposed sensor were in accordance with that of high-performance liquid chromatography-tandem mass spectrometry method. Thus, the developed MIP-based ECL sensor will have a great potential application in rapid and sensitive determination of trace AZM in complex biological samples.