Electrochemically aligned collagen (ELAC) threads fabricated by the isoelectric focusing (IF) of collagen have previously shown potential in tissue engineering and more recently in the fabrication of biohybrid robot structures. For applications in biohybrid robotics, ELAC structures are needed that are both robust and compliant enough to facilitate muscle actuation. However, studies on the effects of IF parameters, and the interactions of such fabrication parameters, on the mechanical and geometric properties of resulting ELAC threads have not been previously found in literature. Understanding the impact of these manufacturing parameters on the material properties is critical to facilitate biohybrid robot design. In this study, the effects of IF duration, IF voltage, and collagen solution concentration were investigated and showed statistically significant effects on adjusting ELAC properties via single-factor experiments. The interactions between parameters exhibited significant joint effects on ELAC property tuning through two-factor experiments. Scanning electron microscopy and 2,4,6-trinitrobenzenesulfonic (TNBS) assays revealed the correlation between high mechanical properties and a combination of low porosity and high degree of crosslinking. By simply tuning IF parameters without changing other fabrication steps, such as crosslinker concentration, ELAC threads with a wide range of mechanical and geometric properties were fabricated. The average tensile modulus of the resulting ELAC threads ranged from 198 ± 90 to 758 ± 138 MPa. The average cross-sectional area ranged from 7756 ± 1000 to 1775 ± 457 . The resultant mapping between IF parameters and ELAC thread properties enabled the production of strong and flexible threads with customizable properties.