Background The architecture of the native enthesis (tendon/bone junction), consists of an organized gradient of tendon, cartilage, and bone tissues. This complex tissue structure effectively transmits the interactions between dynamic muscle tissues and the rigid skeleton. Healed tendon repairs fail to regenerate the appropriate tissue architecture and composition present in the native enthesis. The objective of this study was to create a biologically compatible scaffold that recapitulates the transition of nano-mechanical properties found in the native enthesis. Methods Wild-type mouse Achilles tendon-bone composite tissues were harvested and flash frozen. The modulus of elasticity for each sample was evaluated using atomic force microscopy (AFM). Force curves were generated, and Young’s modulus values calculated using a Hertz-Sneddon model. Biocompatible inks (bio-inks) composed of gelatin methacryloyl (GelMA), and other physiologically relevant additives, such as alginate, hydroxyapatite, and collagen I were fabricated to generate hydrogels of different nano-mechanical properties as assessed using AFM. The gels were crosslinked using Lithium Phenyl (2,4,6-Trimethylbenzoyl) Phosphinate and UV light. The various concentrations of GelMA, additives, and photo cross-linking parameters were then formulated to more closely replicate the transition of nano-mechanical properties found in native enthesis tissues. Results The native mouse enthesis modulus values (n=3) for tendon, cartilage and bone tissues were 111.8 +/- 47.7 MPa, and 36.7 +/- 29.2 MPa, 6.5 +/- 2.6 GPa respectively. Several different formulations of bio-inks were tested to best replicate the modulus values of the native enthesis. Final bio-ink concentrations were created which mimic the nanomechanical properties of tendon, cartilage, and bone. The GelMA concentrations ranged from 10-30%. Hydroxyapatite content ranged from 0-10%. Alginate content ranged from 0-4%. The scaled nanomechanical modulus values for the synthesized hydrogels in each of the different tissue types were 17 +/- 6 KPa, 280 +/- 190 Pa, 87 +/- 28 KPa respectively. Conclusion Three distinct bio-inks were created to form three distinct hydrogels with different concentrations of GelMA and other physiologically-relevant additives. These hydrogels differ in both composition and nano-mechanical properties, analogous to a native enthesis tissue gradient. Future directions include the 3D-printing of these novel bio-inks to create a biomimetic scaffold with a gradient of distinct layers. Each layer will have specific nano-mechanical properties and extracellular matrix components that support the differentiation of mesenchymal progenitor cells into distinct phenotypes reflective of the different tissue types making up the native enthesis.