G-quadruplexes are specialized secondary structures in nucleic acids that possess significant conformational polymorphisms. The precise G-quadruplex conformations in vivo and their relevance to biological functions remain controversial and unclear, especially for telomeric G-quadruplexes. Here, we report a novel single-chain variable fragment (scFv) antibody, D1, with high binding selectivity for parallel G-quadruplexes in vitro and in vivo. Genome-wide chromatin immunoprecipitation using D1 and deep-sequencing revealed the consensus sequence for parallel G-quadruplex formation, which is characterized by G-rich sequence with a short loop size (<3 nt). By using D1, telomeric parallel G-quadruplex was identified and its formation was regulated by small molecular ligands targeting and telomere replication. Together, parallel G-quadruplex specific antibody D1 was found to be a valuable tool for determination of G-quadruplex and its conformation, which will prompt further studies on the structure of G-quadruplex and its biological implication in vivo. Display omitted •A new antibody with parallel G-quadruplex specific recognition•Consensus sequence for parallel G-quadruplex formation on the human genome•Identification of parallel G-quadruplex formation at telomeres•Modulation of telomeric parallel G-quadruplex formation in human cell G-quadruplex conformation in vivo is difficult to assay, especially for telomeric G-quadruplexes. Liu et al. demonstrate the parallel G-quadruplex consensus sequence on the genome and identify the regulated formation of parallel quadruplex at telomeres by using a new antibody.