Stiffness-engineered stretchable substrate technology has been widely used to produce stretchable displays, transistors, and integrated circuits because it is compatible with various flexible electronics technologies. However, the stiffness-engineering technology has never been applied to transistor-based stretchable strain sensors. In this study, we developed thin-film transistor-based strain sensors on stiffness-engineered stretchable substrates. We designed and fabricated strain-sensitive stretchable resistors capable of inducing changes in drain currents of transistors when subjected to stretching forces. The resistors and source electrodes of the transistors were connected in series to integrate the developed stretchable resistors with thin-film transistors on stretchable substrates by printing the resistors after fabricating transistors. The thin-film transistor-based stretchable strain sensors demonstrate feasibility as strain sensors operating under strains of 0%–5%. This strain range can be extended with further investigations. The proposed stiffness-engineering approach will expand the potential for the advancement and manufacturing of innovative stretchable strain sensors.