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Development of sensitive method for assay of proteins secreted from small cell population is important for the assessment of cellular heterogeneity. Here, we developed an ultrasensitive electrochemiluminescence (ECL) aptasensor for secreted protein in small cell population of RAW 264.7 cells for cellular heterogeneity assessment. Tumor necrosis factor-alpha (TNF-α) was chosen as target secreted protein, and specific anti-TNF-α aptamer was chosen as molecule recognition element, while dichlorotris (1,10-phenanthroline) ruthenium(II) hydrate (Ru(phen) 3 2+ ) was chosen as ECL signal compound. An ECL signal probe was prepared by combining aptamer, Ru(phen) 3 2+ , and graphene oxide (GO) through electrostatic interaction and π-π stacking interaction. An aptasensor was fabricated by self-assembly specific aptamer on gold nanoparticles modified electrode for the detection of TNF-α using sandwich assay. High sensitivity and good selectivity were obtained for TNF-α. Parallel analysis was conducted to detect TNF-α secreted by RAW 264.7 cells stimulated by either lipopolysaccharides (LPS) or lupeol for assessing the cell heterogeneity in small cell population and for estimating the intracellular signal transduction in inflammation. Compared to ELISA, our ECL method offers both high sensitivity and good accuracy. The distinct heterogeneity among the secreted protein in small cell population, for the first time, was observed and further correlated to the cell's nature of cluster growth. The developed ECL method incorporating the amplification of nanomaterial and selectivity of aptamer is promising for sensitive and selective analysis of biomolecules in small cell population.