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As a vital enzyme in DNA phosphorylation and restoration, T4 polynucleotide kinase (T4 PNK) has aroused great interest in recent years. Therefore, numerous strategies have been established for highly sensitive detection of T4 PNK based on diverse signal amplification techniques. However, they often need sophisticated design, a variety of auxiliary reagents and enzymes, or cumbersome manipulations. We have designed a new kind of allosteric aptamer probe (AAP) consisting of streptavidin (SA) aptamer and the complementary DNA (cDNA) for simple detection of T4 PNK without signal amplification and with minimized interference in complex biological samples. When the 5′-terminus of the cDNA is phosphorylated by T4 PNK, the cDNA is degraded by lambda exonuclease to release the fluorescein amidite (FAM)-labeled SA aptamer, which subsequently binds to streptavidin beads. The enhancement of the fluorescence signal on SA beads can be detected precisely and easily by a microscope or flow cytometer. Our method performs well in complex biological samples as a result of the enrichment of the signaling molecules on beads, as well as simple manipulations to discard the background interference and nonbinding molecules. Without signal amplification techniques, our AAP method not only avoids complicated manipulations but also decreases the time required. With the advantages of ease of operation, reliability, and robustness for T4 PNK detection in buffer as well as real biological samples, the AAP has great potential for clinical diagnostics, inhibitor screening, and drug discovery.