In the present work, a Smartphone-based Fluorescence Nanoparticle Detector (SPF-NPD) was developed. This method is intended for use in the identification of biological agents in biomedical applications. Here, an android application-based algorithm was developed to analyze the fluorescent nanoparticle intensity level in a target sample. The setup consists of an LED light source, an Eppendorf tube holder, and a smartphone to acquire the fluorescent intensity level in the sample to enable the detection of pathogens within few seconds. High-resolution cameras available on recent smartphones have made live detection more accurate and convenient for healthcare applications. The concept of fluorescent nanoparticle detection with a smartphone has led to a portable device and having potential application in healthcare. In this proposed method the intensity level is analyzed with 5 pixels algorithm, the center pixel followed by four immediate neighbours’ pixels which can analyze with minimal sample quantity. Also, the robustness of the developed algorithm was verified with various megapixel camera ranges from 8 MP to 20 MP.