Background and aim: Decellularized uterine extracellular matrix (dUECM) holds promise for uterine tissue engineering due to its preserved bioactive components and structural integrity. However, bioprinting uterine constructs from dUECM remains unachievable due to the dUECM properties limited for bioprinting. To address issues, this study aimed to (1) develop a one-step protocol to effectively decellularize uterine and (2) synthese dUECM with alginate to form a novel bioactive hydrogel for bioprinting uterine constructs. The in vitro characterization of Alg:dUECM hydrogels with seeded human uterine myometrial-human telomerase reverse transcriptase cells (hTERT-HM) cells has been pursued in terms of mechanical stability, printability and bioactivity for uterine tissue engineering. Materials and Methods: Uterine tissues were decellularized using a one-step protocol with 1% Triton™ X-100 and varying sodium dodecyl sulfate (SDS) concentrations (0.1% - 1.5%) for 48 and 72 hours. Decellularized tissues were analyzed in different forms: fresh samples for tensile testing and histology (DAPI, H&E, Masson's trichrome), freeze-dried samples for SEM imaging, and powdered samples for DNA quantification, glycosaminoglycan (GAG) content, FT-IR, Raman spectroscopy, and TGA. Selected dUECM samples were digested with pepsin and acetic acid, and their rheology and gelation behaviour were assessed. Hydrogels were prepared by combining 2% and 3% alginate with 0.5%, 1%, and 1.5% dUECM. The resulting hydrogels were evaluated for swelling, degradation, mechanical properties, and printability. Biocompatibility was assessed using MTT and Live/Dead assays with hTERT-HM cells seeded on the selected hydrogel. Main Results: The decellularization protocol (1% Triton™ X-100 + 1% SDS for 48 hours) effectively reduced DNA content to 51.33 ± 9.02 ng/mg, nearing the immunogenic threshold of 50 ng/mg, with no visible nuclei, confirming efficient removal of cellular material. Additionally, GAG retention remained high at 54.94 ± 7.55 µg/mg dry weight after 48 hours, comparable to native tissue. FT-IR and Raman spectroscopy analyses confirmed the preservation of collagen's triple-helical structure (Amide III/1450: 1.04 ± 0.03, 1660/1620: 1.32 ± 0.11) with a reduction in thermal stability (TGA peak shift to ≈312°C, peak area 67.18 ± 4.59, p<0.001). In 3D printing studies, 3% Alg + 1.5% dUECM demonstrated the best structural properties, with a balanced printability factor (1.56 ± 0.20), enhanced swelling stability (47 ± 12% at day 14), and superior degradation resistance (94 ± 18% mass retention at day 14). Mechanical analysis confirmed strong structural integrity, with Young’s modulus decreasing from 322.7 ± 50.9 kPa at day 0 to 174.8 ± 29.8 kPa at day 14, outperforming other compositions. Cell viability assays demonstrated that 3% Alg + 1.5% dUECM exhibited a significant hTERT-HM proliferation (258.14 ± 12.83% by day 7, p = 0.0003) compared to 3% Alginate, with enhanced cell attachment. Conclusions: This study demonstrates that the protocol with 1% Triton™ X-100 + 1% SDS can remove cells while preserving ECM components effectively. A hydrogel synthesized from 3% Alg + 1.5% dUECM has appropriate printability, mechanical stability, and able to support cell attachment and proliferation. Collectively, these findings pave the road to engineering uterine tissue for medical applications.