Structural arrangement and dimension play vital roles in wave transport and amplification as they can restrict the volume explored by the waves. However, it is challenging to systematically investigate the interplay among structural, optical, and mechanical properties, in part because of limited experimental platforms that modulate the structural arrangement in a continuous manner. We present light amplification action in Rhodamine B doped silk fibroin (SF) nanofibrous scaffolds and its modulation via the control of the alignment or directionality of SF nanofibers through an electrospinning procedure. Random lasing features of such scaffolds are examined as a function of structural arrangement of the SF nanofibers, and optical-structural-mechanical relationships of the SF-based structures are examined. As SF nanofibers are aligned parallel undergoing a transition from three to quasi-two dimension, light amplification features (e.g., lasing threshold and output power) enhanced, which also strongly correlated with mechanical characteristics (i.e., Young's moduli) of the scaffolds. We confirm such optical characteristics using quasi-mode analyses based on the finite element method. We further demonstrate non-contact, in situ measurement of alternations in lasing features of the scaffolds while the specimens are under tensile loads. These results may highlight potential utility of the scaffolds as a flexible and biocompatible sensor.