On-line solid phase extraction (SPE) coupled to liquid chromatography-mass spectrometry (LC-MS) and biosensors are advanced technologies that have found increasing application in the analysis of environmental contaminants although their application to the determination of emerging contaminants (previously unknown or unrecognized pollutants) has been still limited. This review covers the most recent advances occurred in the areas of on-line SPE-LC-MS and biosensors, discusses and compares the main strengths and limitations of the two approaches, and examines their most relevant applications to the analysis of emerging contaminants in environmental waters. So far, the on-line configuration most frequently used has been SPE coupled to liquid chromatography-(tandem) mass spectrometry. Sorbents used for on-line SPE have included both traditional (alkyl-bonded silicas and polymers) and novel (restricted access materials (RAMs), molecularly imprinted synthetic polymers (MIPs), and immobilized receptors or antibodies (immunosorbents) materials. The biosensor technologies most frequently applied have been based on the use of antibodies and, to a lesser extent, enzymes, bacteria, receptors and DNA as recognition elements, and the use of optical and electrochemical transducing elements. Emerging contaminants investigated by means of these two techniques have included pharmaceuticals, endocrine disrupting compounds such as estrogens, alkylphenols and bisphenol A, pesticides transformation products, disinfection by-products, and bacterial toxins and mycotoxins, among others. Both techniques offer advantageous, and frequently comparable, features such as high sensitivity and selectivity, minimum sample manipulation, and automation. Biosensors are, in addition, relatively cheap and fast, which make them ideally suited for routine testing and screening of samples; however, in most cases, they can not compete yet with on-line SPE procedures in terms of accuracy, reproducibility, reliability (confirmation) of results, and capacity for multi-analyte determination.