Cholesterol is vital for living things because of an important part of cell membranes, as well as one of the basic components of brain and nerve cells. However, cholesterol must be at an optimum level. If it is below or above this level, it is a marker of many various diseases. Therefore, the detection of cholesterol amount is very important. Herein, MWCNTs were synthesized and AgNPs were grown on their surfaces. Thus, AgNP/MWCNT hybrid material was obtained. The AgNP/MWCNTs were used as surface enhancement Raman scattering (SERS) substrate to quantify the cholesterol molecule. High SERS enhancement of AgNP/MWCNT, specific SERS sensing platform and high sensitive SERS substrate were proposed to determine and monitor cholesterol molecule. AgNP/MWCNT substrate was studied in the concentration range of 10−3–10−10 M and the limit of detection (LOD) was calculated as 3.24 × 10−11 M. In addition, the enhancement factor (EF) was determined as 6.21 × 109. As a result, it was reported in this study that cholesterol molecule can be determined with excellent accuracy, precision and sensitive by using AgNP/MWCNT substrate and therefore it can provide great potential in clinical diagnosis and health management.