Second harmonic microscopic imaging and spectroscopy technology has become a powerful tool for biomedical studies, especially in fibrosis-related diseases research. And type I collagen is the major risk factors for fibrotic diseases. In this study, model for three-dimensional (3-D) uniform arrangement type I collagen is set up for researching the second harmonic generation (SHG) on nonlinear optics microscopy. Based on this model, we discuss the influence of different length and size collagen in 3-D arrangement type I collagen. Results can guide us to neatly judge the size, length, and molecules density effect on SHG. For practical application, this theoretical approach can lead us to analyze different severity of collagen diseases. SCANNING 35:12-16, 2013. © 2012 Wiley Periodicals, Inc.