Bimolecular or tetramolecular G-quadruplexes (GQs) are predominantly self-assembled by the same sequence-identical G-rich oligonucleotides and usually remain inert to the strand displacement reaction (SDR) with other short G-rich invading fragments of DNA or RNA. Appealingly, in this study, we demonstrate that a parallel homomeric bimolecular GQ target of Tub10 d(CAGGGAGGGT) as the starting reactant, although completely folded in K+ solution and sufficiently stable (melting temperature of 57.7 °C), can still spontaneously accept strand invasion by a pair of short G-rich invading probes of P1 d(TGGGA) near room temperature. The final SDR product is a novel parallel heteromeric trimolecular GQ (tri-GQ) of Tub10/2P1 reassembled between one Tub10 strand and two P1 strands. Here we present, to the best of our knowledge, the first NMR solution structure of such a discrete heteromeric tri-GQ and unveil a unique mode of two probes vs one target in mutual recognition among G-rich canonical DNA oligomers. As a model system, the short invading probe P1 can spontaneously trap G-rich target Tub10 from a Watson-Crick duplex completely hybridized between Tub10 and its fully complementary strand d(ACCCTCCCTG). The Tub10 sequence of d(CAGGGAGGGT) is a fragment from the G-rich promoter region of the human β2-tubulin gene. Our findings provide new insights into the Hoogsteen pairing-based SDR between a GQ target and double invading probes of short G-rich DNA fragments and are expected to grant access to increasingly complex architectures in GQ-based DNA nanotechnology.