Bicuspid aortic valve (BAV) is an inherited form of heart disease with only two aortic valve leaflets via a disorder of cardiac valvulogenesis. We investigated the in vivo echocardiographic features of cardiac morphology in patients with BAV and the ex vivo compositional components of all the excised BAV leaflets isolated from BAV patients. Three BAV patients were randomly selected. All patients underwent 2D transthoracic echocardiography (TTE) with a Doppler ultrasound tool. The compositional components of each respective BAV leaflet for all the excised BAVs were determined by a portable fiber-optic Raman spectroscopy. Preoperative TTE revealed the thickened and calcified BAV leaflets, and stenotic aortic flow for all BAV patients. These BAV patients exhibited severe aortic stenosis (AS) by the lower values of aortic valve area (AVA) index. One patient showed a more significant left ventricle hypertrophy, whereas two patients exhibited a significant aortic regurgitation (AR). In addition, three different Raman spectral patterns were summed up from 121 randomized Raman determinations for all the excised BAV leaflets. The main calcified deposition in each BAV leaflet was formed by large amounts of calcium hydroxyapatite and type-B carbonate apatite (Raman bands at 960 and 1070 cm-1). The calcified BAV leaflets were composed of different compositional components such as calcium hydroxyapatite, type-B carbonate apatite, lipids, proteins, cholesterol and β-carotene. The rare NL subtype of type 1 BAV morphotype was found in one patient, but two patients had the purely BAV morphotype with two equal-sized leaflets.