The combination of suspension array with rolling circle amplification can lead to a sensitive and specific assay for single-nucleotide polymorphisms (SNPs) detection, as demonstrated in this study. A circular template generated by ligation upon the recognition of a point mutation on DNA targets was amplified isothermally by the Phi29 polymerase on microspheres. The elongation products were labeled with fluorochrome-tagged probes and detected in a flow cytometer, indicating the mutation occurrence. As low as 10 amol of mutated strands was detected by this assay, and positive mutation detection was achieved with a wild-type to mutant ratio of 10 000:1, which could be attributed to the high amplification efficiency of Phi29, the high binding capacity of the microspheres, and the remarkable precision of DNA ligase in distinguishing mismatched bases at the ligation site. A novel design of using two differently labeled detection probes on the same microsphere to target both the wild-type and mutant samples allowed parallel determination of the heterozygosity for two SNPs (K-ras G12C and TP53 R273H) in PCR amplicons prepared from human genomic DNA extracts. This ability lays the groundwork for further enhancing the assay throughput by using multiple fluorophores and microspheres with distinct properties.