Detrusor-sphincter dyssynergia (DSD) represents a core functional impairment of the neurogenic bladder following spinal cord injury; however, no model exists that recapitulates the three-dimensional tissue architecture of the detrusor-sphincter unit and enables analysis of cell–cell communication in vitro. In this study, an innovative spatially defined bioprinting strategy was developed to construct parallel-aligned tissue structures of detrusor smooth muscle cells and urethral sphincter fibroblasts. By precisely controlling the inter-tissue distance to 200 μm, a microenvironment that permits paracrine signaling but prevents direct cell–cell contact was developed. Upon stimulation with inflammatory factors mimicking the pathological state after spinal cord injury, this model enabled, for the first time, visualization and quantitative analysis of the transition of detrusor contractions from synchronous to asynchronous and out-of-phase under the regulation of sphincter-derived paracrine signals. Mechanistic investigations revealed that sphincter fibroblasts specifically upregulate and secrete connective tissue growth factor (CTGF) under pathological conditions, which induces aberrant detrusor contractions via the integrin αvβ3/focal adhesion kinase/extracellular signal-regulated kinase signaling pathway. Neutralizing antibodies against CTGF or the clinical drug mirabegron significantly restored contractile coordination. This study not only provides the first quantifiable in vitro dual-tissue model for DSD research but also uncovers a local inter-tissue communication disorder mechanism independent of neural innervation, opening new avenues for targeted therapy of neurogenic bladder.