Herein, a G-quadruplex-enriched DNA nanonetwork (GDN) self-assembled via Y-modules was designed to construct an ultrasensitive electrochemical biosensing platform with low background for the detection of mucin 1 related to cancers. The single-stranded DNA (ssDNA) S1 converted from target mucin 1 could hybridize with ssDNA S2 and ssDNA S3 with split G-quadruplex fragments at ends to form Y-modules and self-assemble into a GDN, which can capture abundant electroactive substance hemin for a significant electrochemical signal. Impressively, compared with conventional G-quadruplex nanowires with low loading capacity and poor structural stability, the GDN assembled by Y-modules was able to load more signaling probes and obtain a more stable structure for the support of G-quadruplexes, thereby outputting a stronger and more stable electrochemical signal. Moreover, a complete G-quadruplex assembled from two split G-quadruplex effectively reduced the background signal and thus improved the signal-to-noise ratio for the sensitive detection of mucin 1. As a result, the constructed electrochemical biosensor based on GDN achieved ultrasensitive detection of target mucin 1 with a detection limit down to 0.15 fg mL