Biomarkers, including proteins, nucleic acids, and metabolites, are the molecules that can provide insightful information about biological processes and pathological developments. Identification and quantification of biomarkers are highly beneficial for disease diagnosis, progression monitoring, and treatment supervision. However, disease development often involves the complex interplay of molecular networks that limits the utility of individual biomarkers in reaching reliable diagnostic and therapeutic decisions. Thus, recent developments of bioassays have turned the focus to analysis of a collection of biomarkers simultaneously, aiming to improve precision in diagnosis. To achieve the demanded throughput in multiplex detection while keeping the excellent analytical performance in speed, sensitivity, and selectivity, nanomaterials stand out to be the proper enabling tools, with their unique but highly diversified physical and chemical properties and the much advanced synthesis strategies. Herein, this review highlights the recent (2020-2024) developments in the nanomaterial-enabled, optical multiplex sensing techniques. Four key approaches to achieve multiplexity were discussed: spatial coding, signal coding, biocarriers, and data deconvolution using machine learning. We believe these advancements have driven forward the applications of multiplex detection in clinical settings by improving the throughput of biomarker analysis.