The rapid and accurate determination of the dopamine (neurotransmitter) and cholesterol level in bio-fluids is significant because they are crucial bioanalytes for several lethal diseases, which require early diagnosis. The level of DA in the brain is modulated by the dopamine active transporter (DAT), and is influenced by cholesterol levels in the lipid membrane environment. Accordingly, electrochemical biosensors offer rapid and accurate detection and exhibit unique features such as low detection limits even with reduced volumes of analyte, affordability, simple handling, portability and versatility, making them appropriate to deal with augmented challenges in current clinical and point-of-care diagnostics for the determination of dopamine (DA) and cholesterol. This feature article focuses on the development of ultrasensitive electrochemical biosensors for the detection of cholesterol and DA for real-time and onsite applications that can detect targeted analytes with reduced volumes and sub-picomolar concentrations with quick response times. Furthermore, the development of ultrasensitive biosensors via cost-effective, simple fabrication procedures, displaying high sensitivity, selectivity, reliability and good stability is significant in the impending era of electrochemical biosensing. Herein, we emphasize on recent advanced nanomaterials used for the ultrasensitive detection of DA and cholesterol and discuss in depth their electrochemical activities towards ultrasensitive responses. Key points describing future perspectives and the challenges during detection with their probable solutions are discussed, and the current market is also surveyed. Further, a comprehensive review of the literature indicates that there is room for improvement in the miniaturization of cholesterol and dopamine biosensors for lab-on-chip devices and overcoming the current technical limitations to facilitate full utilization by patients at home.